Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Amplified inverted duplications within and adjacent to heterologous selectable DNA.

ABSTRACT: Plasmids containing a dihydrofolate reductase (DHFR) expression unit were transfected into DHFR-deficient Chinese hamster ovary (CHO) cells. Methotrexate exposure was used to select cells with amplified DHFR sequences. Three cell lines were isolated containing amplified copies of transfected DNA that had integrated into the Chinese hamster genome. Plasmid DNA was found to co-amplify with flanking hamster sequences that were repetitive (2 cell lines) and unique (1 cell line). Fragments comprising the junctions of amplified plasmid and CHO DNA were found to exist as inverted duplications in all three cell lines. These observations provide evidence that inverted duplication occurred prior to DNA amplification, thus underscoring the importance of inverted duplication in the DNA amplification process.

SUBMITTER: Heartlein MW

PROVIDER: S-EPMC331829 | biostudies-other | 1989 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Amplified inverted duplications within and adjacent to heterologous selectable DNA.

Heartlein M W MW Latt S A SA

Nucleic acids research 19890201 4

Plasmids containing a dihydrofolate reductase (DHFR) expression unit were transfected into DHFR-deficient Chinese hamster ovary (CHO) cells. Methotrexate exposure was used to select cells with amplified DHFR sequences. Three cell lines were isolated containing amplified copies of transfected DNA that had integrated into the Chinese hamster genome. Plasmid DNA was found to co-amplify with flanking hamster sequences that were repetitive (2 cell lines) and unique (1 cell line). Fragments comprising ...[more]

PMID: 2922290

Similar Datasets

Suppression of expression between adjacent genes within heterologous modules in yeast.

Project description:Recent studies have shown that proximal arrangement of multiple genes can have complex effects on gene expression. For example, in the case of heterologous gene expression modules, certain arrangements of the selection marker and the gene expression cassette may have unintended consequences that limit the predictability and interpretability of module behaviors. The relationship between arrangement and expression has not been systematically characterized within heterologous modules to date. In this study, we quantitatively measured gene expression patterns of the selection marker (KlURA3 driven by the promoter, pKlURA) and the gene expression cassette (GFP driven by the galactose-inducible GAL1 promoter, pGAL1) in all their possible relative arrangements in Saccharomyces cerevisiae. First, we observed that pKlURA activity depends strongly on the relative arrangement and the activity of pGAL1. Most notably, we observed transcriptional suppression in the case of divergent arrangements: pKlURA activity was reduced when pGAL1 was inactive. Based on our nucleosome occupancy data, we attribute the observed transcriptional reduction to nucleosome repositioning. Second, we observed that pGAL1 activity also depends on the relative arrangement of pKlURA. In particular, strains with divergent promoters showed significantly different pGAL1 activation patterns from other strains, but only when their growth was compromised by lack of uracil. We reasoned that this difference in pGAL1 activation patterns arises from arrangement-dependent pKlURA activity that can affect the overall cell physiology (i.e., cell growth and survival in the uracil-depleted condition). Our results underscore the necessity to consider ramifications of promoter arrangement when using synthetic gene expression modules.

| S-EPMC3887525 | biostudies-literature

Inverted duplications on acentric markers: mechanism of formation.

Project description:Acentric inverted duplication (inv dup) markers, the largest group of chromosomal abnormalities with neocentromere formation, are found in patients both with idiopathic mental retardation and with cancer. The mechanism of their formation has been investigated by analyzing the breakpoints and the genotypes of 12 inv dup marker cases (three trisomic, six tetrasomic, two polysomic and one X chromosome derived marker) using a combination of fluorescence in situ hybridization, quantitative SNP array and microsatellite analysis. Inv dup markers were found to form either symmetrically with one breakpoint or asymmetrically with two distinct breakpoints. Genotype analyses revealed that all inv dup markers formed from one single chromatid end. This observation is incompatible with the previously suggested model by which the acentric inv dup markers form through inter-chromosomal U-type exchange. On the basis of the identification of DNA sequence motifs with inverted homologies within all observed breakpoint regions, a new general mechanism is proposed for the acentric inv dup marker formation: following a double-strand break an acentric fragment forms, during either meiosis or mitosis. The open DNA end of the acentric fragment is stabilized by the formation of an intra-chromosomal loop promoted by the presence of sequences with inverted homologies. Likely coinciding with the neocentromere formation, this stabilized fragment is duplicated during an early mitotic event, insuring the marker's survival during cell division and its presence in all cells.

| S-EPMC2685760 | biostudies-literature

Large inverted duplications form via a hairpin mechanism

Project description:Inverted duplications are a common type of copy number variation (CNV) in germline and somatic genomes. Large duplications that include many genes can lead to both neurodevelopmental phenotypes in children and gene amplifications in tumors. There are several models for inverted duplication formation, most of which include a dicentric chromosome intermediate followed by breakage-fusion-bridge (BFB) cycles, but the mechanisms that give rise to the inverted dicentric chromosome in most inverted duplications remain unknown. Here we have combined high-resolution array CGH, custom sequence capture, next-generation sequencing, and long-range PCR to analyze the breakpoints of 50 nonrecurrent inverted duplications in patients with intellectual disability, autism, and congenital anomalies. Sequence analysis of breakpoint junctions reveals a normal-copy disomic spacer between inverted and non-inverted copies of the duplication. Further, short inverted repeats are present at the boundary of the disomic spacer and the inverted duplication. These data support a mechanism of inverted duplication formation whereby a chromosome with a double-strand break intrastrand pairs with itself to form a “hairpin” intermediate that, after DNA replication, produces a dicentric inverted chromosome with a disomic spacer corresponding to the site of the hairpin. We also find evidence of short insertions and inversions at inverted duplication junctions, consistent with a DNA replication-based CNV mechanism. This process can give rise to inverted duplications adjacent to terminal deletions, inverted duplications juxtaposed to translocations, and inverted duplication ring chromosomes

2013-04-03 | GSE45395 | GEO

Large inverted duplications form via a hairpin mechanism

2013-04-03 | E-GEOD-45395 | biostudies-arrayexpress

A familial inverted duplication/deletion of 2p25.1-25.3 provides new clues on the genesis of inverted duplications.

Project description:We studied a family in which the same 10 Mb inverted duplication of 2p25.3-p25.1 segregates in two children and their father, all showing a trisomy phenotype. As FISH analysis demonstrated that the duplication was inverted, we suspected that a contiguous terminal deletion was also present, according to the classical inv dup del type of rearrangements. Although FISH with 2p and 2q subtelomeric probes gave normal results, 100 kb resolution array-C/GH (aCGH) showed that, beside the duplication, a 273 kb deletion was also present. The presence of a single-copy region between the deleted and duplicated regions was further suspected through high-resolution aCGH analysis (approximately 20 kb), although only one informative spot having a normal log ratio was detected. The precise structure of the rearrangement was re-defined by real-time PCR and breakpoint cloning, demonstrating the presence of a 2680 bp single-copy sequence between deleted and duplicated regions and the involvement of a simple repeat with the potential for forming a non-B DNA structure. The rearrangement was not mediated by segmental duplications or short inverted repeats, and the double-strand break might have been repaired by non-homologous end joining or microhomology-mediated intrastrand repair. These data highlight the fact that concomitant deletions associated with inverted duplications are very likely to be more frequent than classical cytogenetic methods alone have been able to demonstrate. The phenotypic effects of the trisomy and of the terminal 2p deletion are discussed.

| S-EPMC2986066 | biostudies-literature

Resolving genomic disorder-associated breakpoints within segmental DNA duplications using massively parallel sequencing.

Project description:The most common recurrent copy-number variants associated with autism, developmental delay and epilepsy are flanked by segmental duplications. Complete genetic characterization of these events is challenging because their breakpoints often occur within high-identity, copy-number polymorphic paralogous sequences that cannot be specifically assayed using hybridization-based methods. Here we provide a protocol for breakpoint resolution with sequence-level precision. Massively parallel sequencing is performed on libraries generated from haplotype-resolved chromosomes, genomic DNA or molecular inversion probe (MIP)-captured breakpoint-informative regions harboring paralog-distinguishing variants. Quantification of sequencing depth over informative sites enables breakpoint localization, typically within several kilobases to tens of kilobases. Depending on the approach used, the sequencing platform, and the accuracy and completeness of the reference genome sequence, this protocol takes from a few days to several months to complete. Once established for a specific genomic disorder, it is possible to process thousands of DNA samples within as little as 3-4 weeks.

| S-EPMC4114152 | biostudies-literature

Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease.

Project description:Extrachromosomal DNA amplification is frequent in the protozoan parasite Leishmania selected for drug resistance. The extrachromosomal amplified DNA is either circular or linear, and is formed at the level of direct or inverted homologous repeated sequences that abound in the Leishmania genome. The RAD51 recombinase plays an important role in circular amplicons formation, but the mechanism by which linear amplicons are formed is unknown. We hypothesized that the Leishmania infantum DNA repair protein MRE11 is required for linear amplicons following rearrangements at the level of inverted repeats. The purified LiMRE11 protein showed both DNA binding and exonuclease activities. Inactivation of the LiMRE11 gene led to parasites with enhanced sensitivity to DNA damaging agents. The MRE11(-/-) parasites had a reduced capacity to form linear amplicons after drug selection, and the reintroduction of an MRE11 allele led to parasites regaining their capacity to generate linear amplicons, but only when MRE11 had an active nuclease activity. These results highlight a novel MRE11-dependent pathway used by Leishmania to amplify portions of its genome to respond to a changing environment.

| S-EPMC4256157 | biostudies-literature

Large inverted duplications in the human genome form via a fold-back mechanism.

Project description:Inverted duplications are a common type of copy number variation (CNV) in germline and somatic genomes. Large duplications that include many genes can lead to both neurodevelopmental phenotypes in children and gene amplifications in tumors. There are several models for inverted duplication formation, most of which include a dicentric chromosome intermediate followed by breakage-fusion-bridge (BFB) cycles, but the mechanisms that give rise to the inverted dicentric chromosome in most inverted duplications remain unknown. Here we have combined high-resolution array CGH, custom sequence capture, next-generation sequencing, and long-range PCR to analyze the breakpoints of 50 nonrecurrent inverted duplications in patients with intellectual disability, autism, and congenital anomalies. For half of the rearrangements in our study, we sequenced at least one breakpoint junction. Sequence analysis of breakpoint junctions reveals a normal-copy disomic spacer between inverted and non-inverted copies of the duplication. Further, short inverted sequences are present at the boundary of the disomic spacer and the inverted duplication. These data support a mechanism of inverted duplication formation whereby a chromosome with a double-strand break intrastrand pairs with itself to form a "fold-back" intermediate that, after DNA replication, produces a dicentric inverted chromosome with a disomic spacer corresponding to the site of the fold-back loop. This process can lead to inverted duplications adjacent to terminal deletions, inverted duplications juxtaposed to translocations, and inverted duplication ring chromosomes.

| S-EPMC3907307 | biostudies-literature

Accelerated exon evolution within primate segmental duplications.

Project description:BackgroundThe identification of signatures of natural selection has long been used as an approach to understanding the unique features of any given species. Genes within segmental duplications are overlooked in most studies of selection due to the limitations of draft nonhuman genome assemblies and to the methodological reliance on accurate gene trees, which are difficult to obtain for duplicated genes.ResultsIn this work, we detected exons with an accumulation of high-quality nucleotide differences between the human assembly and shotgun sequencing reads from single human and macaque individuals. Comparing the observed rates of nucleotide differences between coding exons and their flanking intronic sequences with a likelihood-ratio test, we identified 74 exons with evidence for rapid coding sequence evolution during the evolution of humans and Old World monkeys. Fifty-five percent of rapidly evolving exons were either partially or totally duplicated, which is a significant enrichment of the 6% rate observed across all human coding exons.ConclusionsOur results provide a more comprehensive view of the action of selection upon segmental duplications, which are the most complex regions of our genomes. In light of these findings, we suggest that segmental duplications could be subjected to rapid evolution more frequently than previously thought.

| S-EPMC3906575 | biostudies-literature

CaNAT1, a heterologous dominant selectable marker for transformation of Candida albicans and other pathogenic Candida species.

Project description:A dominant selectable marker for Candida albicans and other Candida species, which confers resistance to nourseothricin, was characterized. In a heterologous promoter system and a recyclable cassette, the marker efficiently permitted deletion and complementation of C. albicans genes. Neither growth nor filamentous development was affected in strains expressing this marker.

| S-EPMC547112 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data