Single-Strand Breaks at Genome Initiate FLT3-ITD Formation

The fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is the most common recurrent mutation in acute myeloid leukemia (AML). FLT3-ITD varies in size from 3 to over 200 bp, resulting in elongation of juxtamembrane domain coded by exon 14 and constitutive kinase activation. The FLT3-ITD is a poor prognostic marker found in 20-30% of AML. However, molecular mechanisms underlying ITD formation are remained to be elucidated. We have analyzed FLT3-ITD mutation-positive AML cases using next-generation sequencing (NGS) and speculated that DNA breakage initiates ITD formation. We developed artificial FLT3-ITD formation assay using CRISPR/Cas9 system. First, genomic DNA from 25 cases with FLT3-ITD mutation-positive AML was used to PCR-amplify the ITD cluster region (ICR; FLT3 exon 14-15) and sequenced by NGS. We extracted more than 3 bp of deletion and insertion. Total 139 independent ITD sequences were identified at varied variant allele frequency (VAF) (0.0005%-45.7%). Each case had 1 to 13 ITDs (median 4 clones). The length of ITDs was 6 to 201 bp (median 48 bp) and 135 (97.1%) unique ITDs showed length with a multiple of 3 bp. In addition, we found 32 clones with Complex ITD, which is considered to have multiple ITD events. Simple FLT3-ITD showed consecutive two repeated sequences with or without filler sequence between the repeated sequences (Fig. Ai). However, some clones showed three or four repetitive sequences (Fig. Aii, Aiii). Furthermore, some clones had sequential second ITD including parts of the first ITD (Fig. Aiv), and some clones had two ITDs at adjacent or distant locations (Fig. Av). These "Complex ITD" was seen in 18 (72%) cases and always accompanying originated "Simple ITD" clones with higher VAF. Total 59 independent deletion sequences were identified in 24 out of 25 (96%) cases. Length of deletion of ICR is 3 to 204 bp (median 4.5 bp). Deletion clone is always rare clone which had a few reads. Non-ITD insertions were found only in 5 clones. The presence of multiple ITD clones in a single case, Complex ITD clones, and deletion clones in the ICR suggest that the ICR is prone to genomic damage, and the mutation process is ongoing in each AML case creating various ITD/deletions. Based on the observation of clinical samples, we investigated whether artificially induced DNA break at ICR repaired as ITDs in the human cell line. The FLT3 ICR was TA-cloned into the pGEM-T easy vector. FLT3 exon 14-targeted guide RNA and Cas9 protein were incubated with the vector in vitro and transfected to HEK293T cells. We compared conventional Cas9 inducing double-strand break (DSB) and Cas9-nickase inducing single-strand break (SSB) to determine the efficacy of ITD formation depending on the different DNA break modes. Genomic DNA was extracted from transfected HEK293T and successfully repaired ICR was amplified with primers annealing to pGEM-T easy vector flanking the cloning site. The amplified PCR product was analyzed by NGS with a 250 bp pair-end read. We extracted 545 and 353 miss repair events from DSB and SSB experiments respectively. The DSB of ICR was repaired as ITDs 1.1%, non-ITD insertions 7.5%, and deletions 91.4% (Fig. B). On the other hand, SSB of ICR was repaired as ITDs 7.3%, non-ITD insertions 1.4%, and deletions 91.2% (Fig. B). Within insertion event, ITD frequency was significantly higher in SSB compared to DSB. (p<0.001; chi-square test). Length of ITDs were 3 to 13 bp (median 3.5 bp) in DSB and 3 to 75 bp (median 28 bp) in SSB experiment (Fig. C). The ITD formed by the SSB was significantly longer than that formed by the DSB (p=0.0013; Mann-Whitney U test) and similar to observation in clinical simple ITD (Fig. C, D). Furthermore, we induced in-vivo SSB at endogenous FLT3 exon14 in HEK293T cells and successfully detected in situ ITDs. Using CRISPR induced SSB, we might develop a cell line with artificial FLT3-ITD which would contribute to deepen understanding of FLT3 biology. SSBs at FLT3 ICR could be a key initiator of FLT3-ITD formation. Progress in understanding the molecular mechanism of FLT3-ITD formation may lead to the development of therapeutic agents in the future. Figure 1 Figure 1. Disclosures Nakagawa: AbbVie GK: Research Funding; Takeda Pharmaceutical Company: Research Funding. Kondo: Astellas Pharma Inc.: Consultancy, Honoraria; Sanwa Kagaku Kenkyusho CO.,LTD: Consultancy; Sumitomo Dainippon Pharma: Honoraria; Bristol-Myers Squibb Company: Honoraria; Novartis Pharma KK: Honoraria; Otsuka Pharmaceutical: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Pfizer: Honoraria. Teshima: Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Company: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Fuji pharma CO.,Ltd: Research Funding; Pfizer Inc.: Honoraria; TEIJIN PHARMA Limited: Research Funding; Gentium/Jazz Pharmaceuticals: Consultancy; Novartis International AG: Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Janssen Pharmaceutical K.K.: Other; Bristol Myers Squibb: Honoraria; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Sanofi S.A.: Research Funding.

Selective Changes in Complexity of Visual Scanning for Social Stimuli in Infancy

In the 1st year of life, infants gradually gain the ability to control their eye movements and explore visual scenes, which support their learning and emerging cognitive skills. These gains include domain-general skills such as rapid orienting or attention disengagement as well as domain-specific ones such as increased sensitivity to social stimuli. However, it remains unknown whether these developmental changes in what infants fixate and for how long in naturalistic scenes lead to the emergence of more complex, repeated sequences of fixations, especially when viewing human figures and faces, and whether these changes are related to improvements in domain-general attentional skills. Here we tested longitudinally the developmental changes in the complexity of fixation sequences at 5.5 and 11 months of age using Recurrence Quantification Analysis. We measured changes in how fixations recur in the same location and changes in the patterns (repeated sequences) of fixations in social and non-social scenes that were either static or dynamic. We found more complex patterns (i.e., repeated and longer sequences) of fixations in social than non-social scenes, both static and dynamic. There was also an age-related increase in the length of repeated fixation sequences only for social static scenes, which was independent of individual differences in orienting and attention disengagement. Our results can be interpreted as evidence for fine-tuning of infants' visual scanning skills. They selectively produce longer and more complex sequences of fixations on faces and bodies before reaching the end of the 1st year of life.

The Formation of Neochromosomes during Experimental Evolution in the Yeast Saccharomyces cerevisiae

Novel, large-scale structural mutations were previously discovered during the cultivation of engineered Saccharomyces cerevisiae strains in which essential tRNA synthetase genes were replaced by their orthologs from the distantly related yeast Yarrowia lipolytica. Among those were internal segmental amplifications forming giant chromosomes as well as complex segmental rearrangements associated with massive amplifications at an unselected short locus. The formation of such novel structures, whose stability is high enough to propagate over multiple generations, involved short repeated sequences dispersed in the genome (as expected), but also novel junctions between unrelated sequences likely triggered by accidental template switching within replication forks. Using the same evolutionary protocol, we now describe yet another type of major structural mutation in the yeast genome, the formation of neochromosomes, with functional centromeres and telomeres, made of extra copies of very long chromosomal segments ligated together in novel arrangements. The novel junctions occurred between short repeated sequences dispersed in the genome. They first resulted in the formation of an instable neochromosome present in a single copy in the diploid cells, followed by its replacement by a shorter, partially palindromic neochromosome present in two copies, whose stability eventually increased the chromosome number of the diploid strains harboring it.

The mitochondrial genomes of bryophytes

In contrast to the highly variable mitogenomes of vascular plants, the composition and architecture of mitogenomes within the three bryophyte lineages appear stable and invariant. Currently, complete mitogenomes are available from 113 bryophyte accessions of 71 genera and 28 orders. Liverworts and mosses hold a rich mitochondrial (mt) gene repertoire among land plants with 40–42 protein-coding genes, whereas hornworts maintain the smallest functional gene set among land plants, of only around two dozen protein-coding genes, with the majority of ribosomal genes pseudogenized and all cytochrome c maturase genes lost. The rRNA and tRNA genes are also conserved and rich in mosses and liverworts, whereas subject to patchy losses in hornworts. In contrast to the conserved gene set, intron content varies significantly with only one intron shared among the three bryophyte lineages. Bryophytes hold relatively compact mitogenomes with narrow size fluctuations. Among the three bryophyte lineages, intergenic spacers and repeat content are smallest in mosses, largest in hornworts, and intermediate in liverworts, mirroring their size differences and levels of structural dynamics among the three lineages. Mosses, with the least repeated sequences, show the most static genome structure; whereas hornworts, with a relatively large set of repeated sequences, experience 1–4 rearrangements; liverworts, with intermediate repeat levels, see only one structural variant that requires two inversions to gain collinearity with the mitogenome of other liverworts. Repeat sequences were evoked to explain the mt gene order rearrangements in hornwort and liverwort mitogenomes; with the latter also supported by sequencing read evidence, which suggests that the conserved mitogenome structure observed in bryophyte lineages might be shaped by low repeat recombination level, and/or along with the intensified nucleus’ surveillance. Mitochondrial RNA editing is abundant in hornworts, with medium frequency and high variation in liverwort species, and generally limited in mosses, reflecting the diversity of nuclear encoded PPR proteins that are functionally related to RNA editing processes.

Discrimination of hospital isolates of Acinetobacter baumannii using repeated sequences and whole genome alignment differential analysis

An optimized method for bacterial strain differentiation, based on combination of Repeated Sequences and Whole Genome Alignment Differential Analysis (RS&WGADA), is presented in this report. In this analysis, 51 Acinetobacter baumannii multidrug-resistance strains from one hospital environment and patients from 14 hospital wards were classified on the basis of polymorphisms of repeated sequences located in CRISPR region, variation in the gene encoding the EmrA-homologue of E. coli, and antibiotic resistance patterns, in combination with three newly identified polymorphic regions in the genomes of A. baumannii clinical isolates. Differential analysis of two similarity matrices between different genotypes and resistance patterns allowed to distinguish three significant correlations (p < 0.05) between 172 bp DNA insertion combined with resistance to chloramphenicol and gentamycin. Interestingly, 45 and 55 bp DNA insertions within the CRISPR region were identified, and combined during analyses with resistance/susceptibility to trimethoprim/sulfamethoxazole. Moreover, 184 or 1374 bp DNA length polymorphisms in the genomic region located upstream of the GTP cyclohydrolase I gene, associated mainly with imipenem susceptibility, was identified. In addition, considerable nucleotide polymorphism of the gene encoding the gamma/tau subunit of DNA polymerase III, an enzyme crucial for bacterial DNA replication, was discovered. The differentiation analysis performed using the above described approach allowed us to monitor the distribution of A. baumannii isolates in different wards of the hospital in the time frame of several years, indicating that the optimized method may be useful in hospital epidemiological studies, particularly in identification of the source of primary infections.

Comparative Distribution of Repetitive Sequences in the Karyotypes of Xenopus tropicalis and Xenopus laevis (Anura, Pipidae)

Xenopus laevis and its diploid relative, Xenopus tropicalis, are the most used amphibian models. Their genomes have been sequenced, and they are emerging as model organisms for research into disease mechanisms. Despite the growing knowledge on their genomes based on data obtained from massive genome sequencing, basic research on repetitive sequences in these species is lacking. This study conducted a comparative analysis of repetitive sequences in X. laevis and X. tropicalis. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) with Cot DNA of both species revealed a conserved enrichment of repetitive sequences at the ends of the chromosomes in these Xenopus species. The repeated sequences located on the short arm of chromosome 3 from X. tropicalis were not related to the sequences on the short arm of chromosomes 3L and 3S from X. laevis, although these chromosomes were homoeologous, indicating that these regions evolved independently in these species. Furthermore, all the other repetitive sequences in X. tropicalis and X. laevis may be species-specific, as they were not revealed in cross-species hybridizations. Painting experiments in X. laevis with chromosome 7 from X. tropicalis revealed shared sequences with the short arm of chromosome 3L. These regions could be related by the presence of the nucleolus organizer region (NOR) in both chromosomes, although the region revealed by chromosome painting in the short arm of chromosome 3L in X. laevis did not correspond to 18S + 28S rDNA sequences, as they did not colocalize. The identification of these repeated sequences is of interest as they provide an explanation to some problems already described in the genome assemblies of these species. Furthermore, the distribution of repetitive DNA in the genomes of X. laevis and X. tropicalis might be a valuable marker to assist us in understanding the genome evolution in a group characterized by numerous polyploidization events coupled with hybridizations.

Transcription termination and antitermination of bacterial CRISPR arrays

A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences (‘repeats’) interspersed with short, variable sequences (‘spacers’). CRISPR arrays are transcribed and processed into individual CRISPR RNAs that each include a single spacer, and direct Cas proteins to complementary sequences in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that Rho can prematurely terminate transcription of bacterial CRISPR arrays, and we identify a widespread antitermination mechanism that antagonizes Rho to facilitate complete transcription of CRISPR arrays. Thus, our data highlight the importance of transcription termination and antitermination in the evolution of bacterial CRISPR-Cas systems.

The Playful Lexicon in the Romance Languages: Prosodic Templates, Onomatopoeia, Reduplication, Clipping, Blending

A lexical item is described as “playful” or “ludic” when it shows evidence of manipulation of the relation that inheres between its form (signifier) and its meaning (signified). The playful lexicon of any given language, therefore, is the sum total of its lexical items that show signs of such manipulation. Linguists have long recognized that the only necessary link between a word’s form and its meaning is the arbitrary social convention that binds them. However, nothing prevents speakers from creating additional, unnecessary and therefore essentially “playful” links, associating forms with meanings in a symbolic, hence non-arbitrary way. This semantic effect is most evident in the case of onomatopoeia, through which the phonetic form of words that designate sounds is designed to be conventionally imitative of the sound. A second group of playful words combines repeated sequences of sounds with meanings that are themselves suggestive of repetition or related concepts such as collectivity, continuity, or actions in sequence, as well as repeated, back-and-forth, or uncontrolled movements, or even, more abstractly, intensity and hesitation. The playfulness of truncated forms such as clips and blends is based on a still more abstract connection between forms and meanings. In the case of clipping, the truncation of the full form of a word triggers a corresponding connotative truncation or diminution of the meaning, that is, a suggestion that the referent is small—either endearingly, humorously, or contemptuously so. In blending, truncation is often accompanied by overlapping, which symbolically highlights the interrelatedness or juxtaposition of the constituents’ individual meanings. Prosodic templates do not constitute a separate category per se; instead, they may play a part in the formation or alteration of words in any of the other categories discussed here.

Marked structural rearrangement of mannose 6-phosphate/IGF2 receptor at different pH environments

Many cell surface receptors internalize their ligands and deliver them to endosomes, where the acidic pH causes the ligand to dissociate. The liberated receptor returns to the cell surface in a process called receptor cycling. The structural basis for pH-dependent ligand dissociation is not well understood. In some receptors, the ligand binding domain is composed of multiple repeated sequences. The insulin-like growth factor 2 receptor (IGF2R) contains 15 β strand–rich repeat domains. The overall structure and the mechanism by which IGF2R binds IGF2 and releases it are unknown. We used cryo-EM to determine the structures of the IGF2R at pH 7.4 with IGF2 bound and at pH 4.5 in the ligand-dissociated state. The results reveal different arrangements of the receptor in different pH environments mediated by changes in the interactions between the repeated sequences. These results have implications for our understanding of ligand release from receptors in endocytic compartments.