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SUMMARY: DNA rearrangements, including inversions, translocations, and large insertions/deletions (indels), are crucial for crop evolution, domestication, and improvement. The rearrangements are frequently induced by ion beams via the mis‐repair of DNA double‐strand breaks (DSBs). Unfortunately, how ion beam‐induced DSBs are repaired has not been comprehensively analyzed and the mechanisms underlying DNA rearrangements remain unclear. In this study, clonal sectors originating from single mutated cells in carbon ion‐irradiated plants were used for whole‐genome sequencing analyses after Arabidopsis seeds and seedlings were irradiated. Comparative analyses of the induced mutations (e.g., size and frequency of indels and microhomology at the junctions of the rearrangements) in the irradiated materials suggested that the broken/rejoined DSB ends were more extensively processed in seedlings than in seeds. A mutation to canonical non‐homologous end‐joining (c‐NHEJ), which is a DSB repair pathway with minimal processing of DSB ends, increased the sensitivity to ion beams more in the seeds than in the seedlings, which was consistent with the junction analysis results, indicative of the minor contribution of c‐NHEJ to the carbon ion‐induced DSB repair in seedlings. Considering the characteristics of the large templated insertions in irradiated seedlings, ion‐beam‐induced DSBs in seedlings are likely repaired primarily by a polymerase theta‐mediated pathway. Polymerase theta‐deficient seedlings were more sensitive to ion beams than the c‐NHEJ‐deficient seedlings, consistent with this hypothesis. This study revealed the key characteristics of ion beam‐induced DSBs and the associated repair mechanisms related to the physiological status of the irradiated materials, with implications for elucidating the occurrence and induction of rearrangements. Significance Statement: DNA rearrangements, which are critical for crop improvement, can be randomly induced in genomes by ion beams. Our genomic analysis of irradiated plants revealed significant differences in the characteristics of the DNA rearrangements in seeds and seedlings, providing important insights into double‐strand break repair mechanisms and the associated rearrangement formation related to the physiological status of the irradiated materials. [ABSTRACT FROM AUTHOR]

Genomic rearrangements contribute to the enhancement of genetic diversity in populations. However, non-canonical rearrangements (NCRs) such as deletions, insertions, and inversions have the potential to trigger genomic instability. In the case of DNA viruses, NCRs can lead to generation of defective viral genomes (DVGs). To study NCRs in herpes simplex virus type 1 (HSV-1) genomes, we enriched DVGs formation by undiluted serial passaging on various cell types. We found that viral passaging on cell type that enables more viral genomes to initiate replication induces higher amplitude and frequency of cyclic patterns associated with DVGs formation. Despite differences in the rates of DVG accumulation, cell lines displayed comparable quantities of distinct NCRs, indicating that fluctuations caused by DVGs may impose bottlenecks on population genetic diversity. These findings propose additional roles for DVGs in modulating viral genetic diversity. Each cell type exhibited a unique population of NCRs, suggesting that NCRs accumulate in a cell type-specific manner. Interestingly, we identified a higher prevalence of short homologies and short reverse complementary in the parental sequences of NCR junction sites across all cell types. These shared sequence characteristics were also observed in NCRs identified in sequences obtained from clinical samples. The fundamental properties of HSV-1 NCR formation uncovered in this study may have broader implications for other DNA viruses. [ABSTRACT FROM AUTHOR]

Abstract A remarkable observation emerging from recent cancer genome analyses is the identification of chromothripsis as a one‐off genomic catastrophe, resulting in massive somatic DNA structural rearrangements (SRs). Largely due to lack of suitable model systems, the mechanistic basis of chromothripsis has remained elusive. We developed an integrative method termed “complex alterations after selection and transformation (CAST),” enabling efficient in vitro generation of complex DNA rearrangements including chromothripsis, using cell perturbations coupled with a strong selection barrier followed by massively parallel sequencing. We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division. Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells. Analysis of primary medulloblastoma cancer genomes verified the link between hyperploidy and chromothripsis in vivo. CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes.

Malignant proliferations are usually clonal. While most times the biological potential can be established through routine pathologic and clinical examinations, some cases are difficult to classify. Moreover, in some situations there are dominant clones whose analysis is important, such as in autoimmune diseases and immunodeficiency. This paper presents in an understandable way the main techniques for the study of clonality, namely: evaluation of gene rearrangements of antigen receptor, and evaluation of human antigen receptor gene.

A remarkable observation emerging from recent cancer genome analyses is the identification of chromothripsis as a one-off genomic catastrophe, resulting in massive somatic DNA structural rearrangements ( SRs). Largely due to lack of suitable model systems, the mechanistic basis of chromothripsis has remained elusive. We developed an integrative method termed 'complex alterations after selection and transformation ( CAST),' enabling efficient in vitro generation of complex DNA rearrangements including chromothripsis, using cell perturbations coupled with a strong selection barrier followed by massively parallel sequencing. We employed this methodology to characterize catastrophic SR formation processes, their temporal sequence, and their impact on gene expression and cell division. Our in vitro system uncovered a propensity of chromothripsis to occur in cells with damaged telomeres, and in particular in hyperploid cells. Analysis of primary medulloblastoma cancer genomes verified the link between hyperploidy and chromothripsis in vivo. CAST provides the foundation for mechanistic dissection of complex DNA rearrangement processes. [ABSTRACT FROM AUTHOR]

As a rule, malignant lymphoid proliferations are clonal. While most of the time the biological potential can be established through routine pathologic examination and auxiliary techniques, some cases are difficult to classify. Moreover, there are situations in which there are dominant clones whose analysis are important, such as occur in autoimmune diseases and immunodeficiency. This paper presents in an understandable way the main techniques for the study of clonality in lymphoid lesions, i.e. the analysis of rearrangements of antigen receptor genes by multiplex polymerase chain reaction (PCR) based tests. [ABSTRACT FROM AUTHOR]

The article presents the study which examined the microsporogenesis in several tomato and barley cultivars infected with various viruses. The aims of the study are to assess the meiotic division in tomato (dicot) and barley (monocot) under viral infection, to identify the meiotic recombination in virus-infected plants and to elucidate the cytogenetic impact of viral infection in different host-pathogen systems. The methods used in the study include the immunosorbent electron microscopy.

Mitochondrial DNA rearrangements occur very frequently in flowering plants and when close to genes there must be concomitant acquisition of new regulatory cis-elements. To explore whether there might be limits to such DNA shuffling, we have mapped the termini of mitochondrial mRNAs in wheat, a monocot, and compared them to the known positions for counterpart genes in the eudicot Arabidopsis. Nine genes share homologous 3′ UTRs over their full-length and for six of them, the termini map very close to the site of wheat/Arabidopsis DNA rearrangements. Only one such case was seen for comparisons of 5′ UTRs, and the 5′ ends of mRNAs are typically more heterogeneous than 3′ termini. Approximately half of the thirty-one wheat mitochondrial transcriptional units are preceded by CRTA promoter-like motifs, and of the potential stem-loop or tRNA-like structures identified as candidate RNA processing/stability signals near the 5′ or 3′ ends, several are shared with Arabidopsis. Comparison of the mitochondrial gene flanking sequences from normal fertile wheat ( Triticum aestivum) with those of Aegilops kotschyi which is the source of mitochondria present in K-type cytoplasmic male sterile wheat, revealed six cases where mRNAs are precluded from sharing full-length homologous UTRs because of genomic reorganization events, and the presence of short repeats located at the sites of discontinuity points to a reciprocal recombination-mediated mode of rearrangement. [ABSTRACT FROM AUTHOR]

n important aspect in the safety assessment of transgenic plants is the exact location of transgene insertion sites within the host genome. However, robust standard operating procedures are not currently available. Using potato as a test species, different methodologies for the determination of insertion sites using a range of published protocols and commercially available kits were assessed in transgenic lines of varying degrees of complexity, from low copy number to complex re-transformed and co-transformed lines. Three commercial kits, APAgene™ GOLD Genome Walking Kit (BIO S&T), DNA Walking SpeedUp™ Kit II (Seegene), and Universal Vectorette™ System (Sigma) were compared with an adaptor-mediated PCR technique. Overall, the APAgene™ kit was used with a high success rate with low copy number potato lines, and also more complex co- and re-transformed lines, and adhering to key confirmation steps it was possible to obtain flanking sequence ranging in size from 300 to 2,500 bp and eliminate PCR artefacts from the analysis. [ABSTRACT FROM AUTHOR]

Genomic disorders are defined as diseases caused by rearrangements of the genome incited by a genomic architecture that conveys instability. Y-chromosome related dysfunctions such as male infertility are frequently associated with gross DNA rearrangements resulting from its peculiar genomic architecture. The Y-chromosome has evolved into a highly specialized chromosome to perform male functions, mainly spermatogenesis. Direct and inverted repeats, some of them palindromes with highly identical nucleotide sequences that can form DNA cruciform structures, characterize the genomic structure of the Y-chromosome long arm. Some particular Y chromosome genomic deletions can cause spermatogenic failure likely because of removal of one or more transcriptional units with a potential role in spermatogenesis. We describe mechanisms underlying the formation of human genomic rearrangements on autosomes and review Y-chromosome deletions associated with male infertility. [ABSTRACT FROM AUTHOR]

Purpose: Chromatin-modifying compounds that inhibit the activity of histone deacetylases have shown potency as radiosensitizers, but the action of these drugs at a molecular level is not clear. Here we investigated the effect of suberoylanilide hydroxyamic acid (SAHA) on DNA breaks and their repair and induction of rearrangements. Methods and Materials: The effect of SAHA on both clonogenic survival and repair was assessed using cell lines SCC-25, MCF7, and TK6. In order to study unique DNA double-strand breaks, anti-CD95 antibody was employed to introduce a DNA double-strand break at a known location within the 11q23 region. The effects of SAHA on DNA cleavage and rearrangements were analyzed by ligation-mediated PCR and inverse PCR, respectively. Results: SAHA acts as radiosensitizer at 1 μM, with dose enhancement factors (DEFs) at 10% survival of: SCC-25 - 1.24 ± 0.05; MCF7 - 1.16 ± 0.09 and TK6 - 1.17 ± 0.05, and it reduced the capacity of SCC-25 cells to repair radiation induced lesions. Additionally, SAHA treatment diffused site-specific fragmentation over at least 1 kbp in TK6 cells. Chromosomal rearrangements produced in TK6 cells exposed to SAHA showed a reduction in microhomology at the breakpoint between 11q23 and partner chromosomes. Conclusions: SAHA shows efficacy as a radiosensitizer at clinically obtainable levels. In its presence, targeted DNA strand breaks occur over an expanded region, indicating increased chromatin access. The rejoining of such breaks is degraded by SAHA when measured as rearrangements at the molecular level and rejoining that contributes to cell survival. [Copyright &y& Elsevier]

Abstract: Motivated by DNA rearrangements and DNA homologous recombination modeled in [A. Angeleska, N. Jonoska, M. Saito, L.F. Landweber, RNA-guided DNA assembly, Journal of Theoretical Biology, 248(4) (2007), 706–720], we investigate smoothings on graphs that consist of only 4-valent and 1-valent rigid vertices, called assembly graphs. An assembly graph can be seen as a representation of the DNA during certain recombination processes in which 4-valent vertices correspond to the alignment of the recombination sites. A single gene is modeled by a polygonal path in an assembly graph. A polygonal path makes a “right-angle” turn at every vertex, defining smoothings at the 4-valent vertices and therefore modeling the recombination process. We investigate the minimal number of polygonal paths visiting all vertices of a given graph exactly once, and show that for every positive integer there are graphs that require at least such polygonal paths. We show that there is an embedding in three-dimensional space of each assembly graph such that smoothing of vertices according to a given set of polygonal paths results in an unlinked graph. As some recombination processes may happen simultaneously, we characterize the subsets of vertices whose simultaneous smoothings keep a given gene in tact and give a characterization of all sequences of sets of vertices defining successive simultaneous smoothings that can realize complete gene rearrangement. [Copyright &y& Elsevier]

Drugs that target DNA topoisomerase II (Top2), including etoposide (VP-16), doxorubicin, and mitoxantrone, are among the most effective anticancer drugs in clinical use. However, Top2-based chemotherapy has been associated with higher incidences of secondary malignancies, notably the development of acute myeloid leukemia in VP-16-treated patients. This association is suggestive of a link between carcinogenesis and Top2-mediated DNA damage. We show here that VP-16-induced carcinogenesis involves mainly the β rather than the a isozyme of Top2. In a mouse skin carcinogenesis model, the incidence of VP-16-induced melanomas in the skin of 7,12-dimethylbenz[a]anthracene-treated mice is found to be significantly higher in TOP2β+ than in skin-specific top2β-knockout mice. Furthermore, VP-16-induced DNA sequence rearrangements and double-strand breaks (DSBs) are found to be Top2β-dependent and preventable by cotreatment with a proteasome inhibitor, suggesting the importance of proteasomal degradation of the Top2β-DNA cleavage complexes in VP-16-induced DNA sequence rearrangements. VP-16 cytotoxicity in transformed cells expressing both Top2 isozymes is, however, found to be primarily Top2α-dependent. These results point to the importance of developing Top2α-specific anticancer drugs for effective chemotherapy without the development of treatment-related secondary malignancies. [ABSTRACT FROM AUTHOR]

Genetic recombination in bacteria is facilitated by the RecA strand transfer protein and strongly depends on the homology between interacting sequences. RecA-independent recombination is detectable but occurs at extremely low frequencies and is less responsive to the extent of homology. In this article, we show that RecA-independent recombination in Escherichia coil is depressed by the redundant action of single-strand exonucleases. In the absence of multiple single-strand exonucleases, the efficiency of RecA-independent recombination events, involving either gene conversion or crossing-over, is markedly increased to levels rivaling RecA-dependent events. This finding suggests that RecA-independent recombination is not intrinsically inefficient but is limited by single-strand DNA substrate availability. Crossing-over is inhibited by exonucleases ExoI, ExoVII, ExoX, and RecJ, whereas only ExoI and RecJ abort gene-conversion events. In ExoI¯ RecJ¯ strains, gene conversion can be accomplished by transformation of short single-strand DNA oligonucleotides and is more efficient when the oligonucleotide is complementary to the lagging-strand replication template. We propose that E. coil encodes an unknown mechanism for RecA-independent recombination (independent of prophage recombination systems) that is targeted to replication forks. The potential of RecA-independent recombination to mediate exchange at short homologies suggests that it may contribute significantly to genomic change in bacteria, especially in species with reduced cellular exonuclease activity or those that encode DNA protection factors. [ABSTRACT FROM AUTHOR]

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G>A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 ( n=2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 ( n=2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies. [ABSTRACT FROM AUTHOR]

P transposable elements in Drosophila are members of a larger class of mobile elements that move using a cut-and-paste mechanism. P-element transposase uses guanosine triphosphate (GTP) as a cofactor for transposition. Here, we use atomic force microscopy (AFM) to visualize protein-DNA complexes formed during the initial stages of P-element transposition. These studies reveal that GTP acts to promote assembly of the first detectable noncovalent precleavage synaptic complex. This initial complex then randomly and independently cleaves each P-element end. These data show that GTP acts to promote protein-DNA assembly, and may explain why P-element excision often leads to unidirectional deletions. [ABSTRACT FROM AUTHOR]

Physicists question whether there are ‘universals’ in biology. One reason is that the prevailing theory of biological evolution postulates a random walk to each new adaptation. In the last 50 years, molecular genetics has revealed features of DNA sequence organization, protein structure and cellular processes of genetic change that suggest evolution by Natural Genetic Engineering. Genomes are hierarchically organized as systems assembled from DNA modules. Each genome is formatted and integrated by repetitive DNA sequence elements that do not code for proteins, much as a computer drive is formatted. These formatting elements constitute codons in multiple genetic codes for distinct functions such as transcription, replication, DNA compaction and genome distribution to daughter cells. Consequently, there is a computation-ready Genome System Architecture for each species. Whole-genome sequencing indicates that rearrangement of genetic modules plus duplication and reuse of existing genomic systems are fundamental events in evolution. Studies of genetic change show that cells possess mobile genetic elements and other natural genetic engineering activities to carry out the necessary DNA reorganizations. Natural genetic engineering functions are sensitive to biological inputs and their non-random operations help explain how novel genome system architectures can arise in evolution. [ABSTRACT FROM AUTHOR]

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process. [ABSTRACT FROM AUTHOR]

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are two dominantly inherited disorders affecting the peripheral nervous system. The relationship between these two disorders is unique among related syndromes. The tandem duplication of a 1.5 Mb region located on chromosome 17 (17p11.2-p12), which contains the peripheral myelin protein gene PMP22, results in a CMT1A phenotype, whereas the reciprocal deletion of this 1.5 Mb region yields an HNPP phenotype. Therefore, CMT1A duplication patients are triploid for this region of chromosome 17, and HNPP deletion patients are haploid. The genetic mechanism that generates the phenotypic features in aneuploid mental retardation syndromes like Down syndrome (trisomy 21) and Miller-Dieker syndrome (del 17p13) may prove to be similar to the PMP22 gene dosage effects, which result in CMT1A and HNPP. Studying the relationship between the phenotypic features and the underlying mechanism of these two neuropathies may elucidate unique features of neuronal genes that predispose them to dosage sensitivity. [ABSTRACT FROM AUTHOR]

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E. coli. [ABSTRACT FROM AUTHOR]

The surfaces of Tetrahymena thermophila cells grown between 20 and 35 °C are covered by one or more variants of H antigens. A cDNA clone, pC6, has previously been identified that hybridizes to a unique polyA RNA that appears to code for the SerH3 variant of the H antigens. pC6 and a subclone of it, pGpC6.295, were used to analyze the genomic organization of the corresponding gene(s) in both the macronucleus and the micronucleus. It was determined that pC6 hybridizes to a small family of sequences in the macronucleus, only one of which also hybridizes to pGpC6.295. The latter is a strong candidate for the gene encoding the SerH3 antigen. Sequences homologous to pC6 - but not to pGpC6.295 - are present in strains carrying the other SerH alleles. Shifts in antigen switching during vegetative growth do not result in any detectable DNA rearrangements in the vicinity of the pC6-hybridizing sequence family. Analysis of micronuclear DNA from a homozygous SerH3 strain revealed that it also contains a family of sequences that are homologous to pC6; but, in contrast to the macronuclear DNA, two members of this micronuclear sequence family hybridize to pGpC6.295. Comparison of micro- and macronuclear DNA indicate that some members of the pC6-positive sequence family rearrange during macronuclear development. These rearrangements fall into two classes: those which occur reproducibly, and those which show variability. The gene homologous to pGp6.295 falls into the former category. [ABSTRACT FROM AUTHOR]

Two genes, designated Trsc-UAA and Trsc-UBA, which encode highly polymorphic major histocompatibility complex (MHC) class I molecules in the shark Triakis scyllia were isolated. The identification of these genes indicates that the classical MHC class I was already established at the level of elasmobranchs during animal evolution. At the emergence of the MHC/T cell receptor recognition system, the number of genes for T cell receptors (TCR) must have been just one. In this brief review, the way in which a small number of TCR could have recognized MHC-oligopeptide complexes initially, based on recent progress in the phylogenetic analysis of the immune systems in primitive vertebrates, is discussed. [ABSTRACT FROM AUTHOR]