Amplicon rearrangements during the extrachromosomal and intrachromosomal amplification process in a glioma

The mechanisms of gene amplification in tumour cells are poorly understood and the relationship between extrachromosomal DNA molecules, named double minutes (dmins), and intrachromosomal homogeneously staining regions (hsr) is not documented at nucleotide resolution. Using fluorescent in situ hybridization and whole genome sequencing, we studied a xenografted human oligodendroglioma where the co-amplification of the EGFR and MYC loci was present in the form of dmins at early passages and of an hsr at later passages. The amplified regions underwent multiple rearrangements and deletions during the formation of the dmins and their transformation into hsr. In both forms of amplification, non-homologous end-joining and microhomology-mediated end-joining rather than replication repair mechanisms prevailed in fusions. Small fragments, some of a few tens of base pairs, were associated in contigs. They came from clusters of breakpoints localized hundreds of kilobases apart in the amplified regions. The characteristics of some pairs of junctions suggest that at least some fragments were not fused randomly but could result from the concomitant repair of neighbouring breakpoints during the interaction of remote DNA sequences. This characterization at nucleotide resolution of the transition between extra- and intrachromosome amplifications highlights a hitherto uncharacterized organization of the amplified regions suggesting the involvement of new mechanisms in their formation.     

DNA structural features on borders of ERBB2 amplicons in breast cancer

In 25–30% of cases of breast cancer tumors, the amplification of the chromosome fragment around ERBB2 underlies the increased expression of genes adjacent to ERBB2. The increased expression of genes within ERBB2-containing amplicons may impact not only the growth and development of the tumor, but also the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of the amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions during intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by the structural peculiarities of DNA, rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of ten genes adjacent to ERBB2 was evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFN1A3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with a high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. Based on the obtained results, one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification.     

Increase in incidence of chromosome instability and non-conservative recombination between repeats in Saccharomyces cerevisiae hpr1Δ strains

Null hprl Δ strains show a large increase (up to 2000-fold) over wild type in the frequency of occurrence of deletions between direct repeats on three different chromosomes. However, we show that hprl Δ mutations have little or no effect on reciprocal exchange, gene conversion or unequal sister chromatid exchange, as determined using intrachromosomal, interchromosomal and plasmid-chromosome assay systems. A novel intrachromosomal recombination system has allowed us to determine that over 95% of deletions in hpr1 Δ strains do not occur by reciprocal exchange. On the other hand, hpr1 Δ strains show chromosome loss frequencies of up to 100 times the wild-type level. Our results suggest that yeast cells have a very efficient non-conservative recombination mechanism, dependent on RADI and RAD52, that causes deletions between direct DNA repeats, and this mechanism is strongly stimulated in hpr1 Δ strains. The results indicate that the Hpr1 protein is required for stability of DNA repeats and chromosomes. We propose that in the absence of the Hprl protein the cell destabilizes the genome by allowing the initiation of events that lead to deletions of sequences between repeats, and to chromosome instability. We discuss the roles that proteins such as Hprl have in maintaining direct repeats and in preventing non-conservative recombination and consider the importance of these functions for chromosome stability.     

A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae

Gene amplifications have been detected as a transitory phenomenon in bacterial cultures. They are predicted to contribute to rapid adaptation by simultaneously increasing the expression of genes clustered on the chromosome. However, genome amplifications have rarely been described in natural isolates. Through DNA array analysis, we have identified two Streptococcus agalactiae strains carrying tandem genome amplifications: a fourfold amplification of 13.5 kb and a duplication of 92 kb. Both amplifications were located close to the terminus of replication and originated independently from any long repeated sequence. They probably arose in the human host and showed different stabilities, the 13.5-kb amplification being lost at a frequency of 0.003 per generation and the 92-kb tandem duplication at a frequency of 0.035 per generation. The 13.5-kb tandem amplification carried the five genes required for dihydrofolate biosynthesis and led to both trimethoprim (TMP) and sulfonamide (SU) resistance. Resistance to SU probably resulted from the increased synthesis of dihydropteroate synthase, the target of this antibiotic, whereas the amplification of the whole pathway was responsible for TMP resistance. This revealed a new mechanism of resistance to TMP involving an increased dihydrofolate biosynthesis. This is, to our knowledge, the first reported case of naturally occurring antibiotic resistance resulting from genome amplification in bacteria. The low stability of DNA segment amplifications suggests that their role in antibiotic resistance might have been underestimated.     

Multiple displacement amplification of the DNA from single flow–sorted plant chromosome

A protocol is described for production of micrograms of DNA from single copies of flow‐sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome‐derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single‐chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome‐specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole‐genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.     

DNA amplifications and deletions in Streptomyces lividans 66 and the loss of one end of the linear chromosome

Thirty-two 2-deoxygalactose-resistant mutants with DNA amplifications were isolated from Streptomyces lividans 66 strains carrying plasmid pMT664, which carries an agarase gene (dagA) and IS466. Thirty-one of the mutants carried amplified DNA sequences from a 70 kb region about 300 kb from one end of the linear chromosome in this species. In 28 of the mutants, all the wild-type sequences between the amplified region and the start of the 30 kb inverted repeat that forms the chromosome end were deleted. Thus, there appeared to be loss of one chromosome end and its replacement by the DNA amplification. In some mutants there amplification of a previously characterised 5.7 kb sequence that lies about 600 kb from the other chromosome end was also noted.     

B chromosomes of the Podisma sapporensis Shir. (Orthoptera, Acrididae) analysed by chromosome microdissection and FISH

The analysis of the distribution of repetitive DNA of the B chromosomes of Podisma sapporensis in the A and B chromosomes of the natural populations and in A chromosomes of three other species of the Podismini grasshoppers were made. DNA-libraries of the B chromosome and the euchromatic segment of the A chromosome of P. sapporensis were generated by meiotic chromosome microdissection followed by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). Paints based on these DNA-libraries were used for FISH analysis to detect localization of homologous sequences in A and B chromosomes of P. sapporensis from different natural populations. On the basis of the FISH analysis the authors suggest that evolution of the B chromosomes in Podisma sapporensis was associated mainly with the insertions of "alien DNA sequences" into ancestral A chromosome and their further amplification. The number of initial sites of amplifications differed in the different Bs, the distance between these sites also varying. Karyotype evolution in P. sapporensis was associated partly with the insertion of "alien DNA sequences" into pericentromeric chromosomal regions. Insertion into the small short arms of the acrocentric chromosomes followed, with the DNA amplification leading to the formation of the additional C-heterochromatic arms or euchromatic-like regions of different size.     

Single Origin of Sex Chromosomes and Multiple Origins of B Chromosomes in Fish Genus Characidium

Chromosome painting with DNA probes obtained from supernumerary (B) and sex chromosomes in three species of fish genus Characidium (C. gomesi, C. pterostictum and C. oiticicai) showed a close resemblance in repetitive DNA content between B and sex chromosomes in C. gomesi and C. pterostictum. This suggests an intraspecific origin for B chromosomes in these two species, probably deriving from sex chromosomes. In C. oiticicai, however, a DNA probe obtained from its B chromosome hybridized with the B but not with the A chromosomes, suggesting that the B chromosome in this species could have arisen interspecifically, although this hypothesis needs further investigation. A molecular phylogenetic analysis performed on nine Characidium species, with two mtDNA genes, showed that the presence of heteromorphic sex chromosomes in these species is a derived condition, and that their origin could have been unique, a conclusion also supported by interspecific chromosome painting with a CgW probe derived from the W chromosome in C. gomesi. Summing up, our results indicate that whereas heteromorphic sex chromosomes in the genus Characidium appear to have had a common and unique origin, B chromosomes may have had independent origins in different species. Our results also show that molecular phylogenetic analysis is an excellent complement for cytogenetic studies by unveiling the direction of evolutionary chromosome changes.     

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.     

Asynchronous Replication,Mono-Allelic Expression,and Long Range Cis-Effects of ASAR6

Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.     

Highly Differentiated ZW Sex Microchromosomes in the Australian Varanus Species Evolved through Rapid Amplification of Repetitive Sequences

Transitions between sex determination systems have occurred in many lineages of squamates and it follows that novel sex chromosomes will also have arisen multiple times. The formation of sex chromosomes may be reinforced by inhibition of recombination and the accumulation of repetitive DNA sequences. The karyotypes of monitor lizards are known to be highly conserved yet the sex chromosomes in this family have not been fully investigated. Here, we compare male and female karyotypes of three Australian monitor lizards, Varanus acanthurus, V. gouldii and V. rosenbergi, from two different clades. V. acanthurus belongs to the acanthurus clade and the other two belong to the gouldii clade. We applied C-banding and comparative genomic hybridization to reveal that these species have ZZ/ZW sex micro-chromosomes in which the W chromosome is highly differentiated from the Z chromosome. In combination with previous reports, all six Varanus species in which sex chromosomes have been identified have ZZ/ZW sex chromosomes, spanning several clades on the varanid phylogeny, making it likely that the ZZ/ZW sex chromosome is ancestral for this family. However, repetitive sequences of these ZW chromosome pairs differed among species. In particular, an (AAT)n microsatellite repeat motif mapped by fluorescence in situ hybridization on part of W chromosome in V. acanthurus only, whereas a (CGG)n motif mapped onto the W chromosomes of V. gouldii and V. rosenbergi. Furthermore, the W chromosome probe for V. acanthurus produced hybridization signals only on the centromeric regions of W chromosomes of the other two species. These results suggest that the W chromosome sequences were not conserved between gouldii and acanthurus clades and that these repetitive sequences have been amplified rapidly and independently on the W chromosome of the two clades after their divergence.     

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.     

Delimiting the Origin of a B Chromosome by FISH Mapping,Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei,Characiformes)

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.     

Structural analysis of aberrant chromosomes that occur spontaneously in diploid Saccharomyces cerevisiae: retrotransposon Ty1 plays a crucial role in chromosomal rearrangements.

The structural analysis of aberrant chromosomes is important for our understanding of the molecular mechanisms underlying chromosomal rearrangements. We have identified a number of diploid Saccharomyces cerevisiae clones that have undergone loss of heterozygosity (LOH) leading to functional inactivation of the hemizygous URA3 marker placed on the right arm of chromosome III. Aberrant-sized chromosomes derived from chromosome III were detected in approximately 8% of LOH clones. Here, we have analyzed the structure of the aberrant chromosomes in 45 LOH clones with a PCR-based method that determines the ploidy of a series of loci on chromosome III. The alterations included various deletions and amplifications. Sequencing of the junctions revealed that all the breakpoints had been made within repeat sequences in the yeast genome, namely, MAT-HMR, which resulted in intrachromosomal deletion, and retrotransposon Ty1 elements, which were involved in various translocations. Although the translocations involved different breakpoints on different chromosomes, all breakpoints were exclusively within Ty1 elements. Some of the resulting Ty1 elements left at the breakpoints had a complex construction that indicated the involvement of other Ty1 elements not present at the parental breakpoints. These indicate that Ty1 elements are crucially involved in the generation of chromosomal rearrangements in diploid yeast cells.     

Normal Segregation of a Foreign-Species Chromosome During Drosophila Female Meiosis Despite Extensive Heterochromatin Divergence

The abundance and composition of heterochromatin changes rapidly between species and contributes to hybrid incompatibility and reproductive isolation. Heterochromatin differences may also destabilize chromosome segregation and cause meiotic drive, the non-Mendelian segregation of homologous chromosomes. Here we use a range of genetic and cytological assays to examine the meiotic properties of a Drosophila simulans chromosome 4 (sim-IV) introgressed into D. melanogaster. These two species differ by ∼12–13% at synonymous sites and several genes essential for chromosome segregation have experienced recurrent adaptive evolution since their divergence. Furthermore, their chromosome 4s are visibly different due to heterochromatin divergence, including in the AATAT pericentromeric satellite DNA. We find a visible imbalance in the positioning of the two chromosome 4s in sim-IV/mel-IV heterozygote and also replicate this finding with a D. melanogaster 4 containing a heterochromatic deletion. These results demonstrate that heterochromatin abundance can have a visible effect on chromosome positioning during meiosis. Despite this effect, however, we find that sim-IV segregates normally in both diplo and triplo 4 D. melanogaster females and does not experience elevated nondisjunction. We conclude that segregation abnormalities and a high level of meiotic drive are not inevitable byproducts of extensive heterochromatin divergence. Animal chromosomes typically contain large amounts of noncoding repetitive DNA that nevertheless varies widely between species. This variation may potentially induce non-Mendelian transmission of chromosomes. We have examined the meiotic properties and transmission of a highly diverged chromosome 4 from a foreign species within the fruitfly Drosophila melanogaster. This chromosome has substantially less of a simple sequence repeat than does D. melanogaster 4, and we find that this difference results in altered positioning when chromosomes align during meiosis. Yet this foreign chromosome segregates at normal frequencies, demonstrating that chromosome segregation can be robust to major differences in repetitive DNA abundance.     

Sex chromosome system ZZ/ZW in Apareiodon hasemani Eigenmann, 1916 (Characiformes,Parodontidae) and a derived chromosomal region

Parodontidae fish show few morphological characteristics for the identification of their representatives and chromosomal analyses have provided reliable features for determining the interrelationships in this family. In this study, the chromosomes of Apareiodon hasemani from the São Francisco River basin, Brazil, were analyzed and showed a karyotype with 2n = 54 meta/submetacentric chromosomes, and a ZZ/ZW sex chromosome system. The study revealed active NORs located on pair 11 and additional 18S rDNA sites on pairs 7 and 22. The 5S rDNA locus was found in pair 14. It showed a pericentric inversion regarding the ancestral condition. The satellite DNA pPh2004 was absent in the chromosomes of A. hasemani, a shared condition with most members of Apareiodon. The WAp probe was able to detect the amplification region of the W chromosome, corroborating the common origin of the system within Parodontidae. These chromosomal data corroborate an origin for the ZW system of Parodontidae and aid in the understanding of the differentiation of sex chromosome systems in Neotropical fishes.