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1.
Gene ; 767: 145186, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32998045

RESUMO

In ciliates, with every sexual event the transcriptionally active genes of the sub-chromosomic somatic genome that resides in the cell macronucleus are lost. They are de novo assembled starting from 'Macronuclear Destined Sequences' that arise from the fragmentation of transcriptionally silent DNA sequences of the germline chromosomic genome enclosed in the cell micronucleus. The RNA-mediated epigenetic mechanism that drives the assembly of these sequences is subject to errors which result in the formation of chimeric genes. Studying a gene family that in Euplotes raikovi controls the synthesis of protein signal pheromones responsible for a self/not-self recognition mechanism, we identified the chimeric structure of an 851-bp macronuclear gene previously known to specify soluble and membrane-bound pheromone molecules through an intron-splicing mechanism. This chimeric gene, designated mac-er-1*, conserved the native pheromone-gene structure throughout its coding and 3' regions. Instead, its 5' region is completely unrelated to the pheromone gene structure at the level of a 360-bp sequence, which derives from the assembly with a MDS destined to compound a 2417-bp gene encoding a 696-amino acid protein with unknown function. This mac-er-1* gene characterization provides further evidence that ciliates rely on functional chimeric genes that originate in non-programmed phenomena of somatic MDS recombination to increase the species genetic variability independently of gene reshuffling phenomena of the germline genome.


Assuntos
Quimera/genética , Euplotes/genética , Feromônios/genética , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Cilióforos/genética , DNA/genética , Rearranjo Gênico/genética , Íntrons/genética , RNA/genética , Processamento de RNA/genética
2.
PLoS Genet ; 16(7): e1008949, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32702045

RESUMO

In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements.


Assuntos
Núcleo Celular/genética , Chaperonas de Histonas/genética , Paramecium/genética , Transposases/genética , Sequência de Bases/genética , Quebras de DNA de Cadeia Dupla , Clivagem do DNA , DNA de Protozoário/genética , Endonucleases , Rearranjo Gênico/genética , Genoma de Protozoário/genética , Paramecium/crescimento & desenvolvimento
3.
Cell Mol Life Sci ; 77(22): 4615-4629, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32462406

RESUMO

Ciliates are a highly divergent group of unicellular eukaryotes with separate somatic and germline genomes found in distinct dimorphic nuclei. This characteristic feature is tightly linked to extremely laborious developmentally regulated genome rearrangements in the development of a new somatic genome/nuclei following sex. The transformation from germline to soma genome involves massive DNA elimination mediated by non-coding RNAs, chromosome fragmentation, as well as DNA amplification. In this review, we discuss the similarities and differences in the genome reorganization processes of the model ciliates Paramecium and Tetrahymena (class Oligohymenophorea), and the distantly related Euplotes, Stylonychia, and Oxytricha (class Spirotrichea).


Assuntos
Cilióforos/genética , Rearranjo Gênico/genética , Genoma de Protozoário/genética , Animais , Núcleo Celular/genética , Células Germinativas/fisiologia , Humanos
4.
Virchows Arch ; 477(1): 33-45, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32447492

RESUMO

Biliary tract carcinomas are divided into intrahepatic, perihilar, distal extrahepatic cholangiocarcinomas, and gallbladder adenocarcinomas. Therapies targeting ROS1, ALK, MET, and HER2 alterations are currently evaluated in clinical trials. We assessed ROS1 and ALK translocations/amplifications as well as MET and HER2 amplifications for each tumor subtype by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) in 73 intrahepatic, 40 perihilar bile duct, 36 distal extrahepatic cholangiocarcinomas, and 45 gallbladder adenocarcinomas (n = 194). By FISH, we detected targetable alterations in 5.2% of cases (n = 10): HER2 and MET amplifications were found in 4.1% (n = 8) and 1.0% (n = 2), respectively. The HER2-amplified cases were mostly gallbladder adenocarcinomas (n = 5). The MET- and HER2-amplified cases were all positive by IHC. Fourteen cases without MET amplification were positive by IHC, whereas HER2 over-expression was detected by IHC only in HER2-amplified cases. We detected no ALK or ROS1 translocation or amplification. Several alterations were consistent with aneuploidy: 24 cases showed only one copy of ROS1 gene, 4 cases displayed a profile of chromosomal instability, and an over-representation of centromeric alpha-satellite sequences was found in five cases. We confirm a relatively high rate of HER2 amplifications in gallbladder adenocarcinomas and the efficacy of IHC to screen these cases. Our results also suggest the value of IHC to screen MET amplification. Contrary to initial publications, ROS1 rearrangements seem to be very rare in biliary tract adenocarcinomas. We confirm a relatively high frequency of aneuploidy and chromosomal instability and reveal the over-representation of centromeric alpha-satellite sequences in intrahepatic cholangiocarcinomas.


Assuntos
Adenocarcinoma/genética , Rearranjo Gênico/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptor ErbB-2/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Quinase do Linfoma Anaplásico/genética , Sistema Biliar/patologia , Humanos , Hibridização in Situ Fluorescente/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-met/genética
5.
Proc Natl Acad Sci U S A ; 117(18): 9912-9921, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32321829

RESUMO

Triple-negative breast cancer (TNBC) accounts for 10 to 20% of breast cancer, with chemotherapy as its mainstay of treatment due to lack of well-defined targets, and recent genomic sequencing studies have revealed a paucity of TNBC-specific mutations. Recurrent gene fusions comprise a class of viable genetic targets in solid tumors; however, their role in breast cancer remains underappreciated due to the complexity of genomic rearrangements in this cancer. Our interrogation of the whole-genome sequencing data for 215 breast tumors catalogued 99 recurrent gene fusions, 57% of which are cryptic adjacent gene rearrangements (AGRs). The most frequent AGRs, BCL2L14-ETV6, TTC6-MIPOL1, ESR1-CCDC170, and AKAP8-BRD4, were preferentially found in the more aggressive forms of breast cancers that lack well-defined genetic targets. Among these, BCL2L14-ETV6 was exclusively detected in TNBC, and interrogation of four independent patient cohorts detected BCL2L14-ETV6 in 4.4 to 12.2% of TNBC tumors. Interestingly, these fusion-positive tumors exhibit more aggressive histopathological features, such as gross necrosis and high tumor grade. Amid TNBC subtypes, BCL2L14-ETV6 is most frequently detected in the mesenchymal entity, accounting for ∼19% of these tumors. Ectopic expression of BCL2L14-ETV6 fusions induce distinct expression changes from wild-type ETV6 and enhance cell motility and invasiveness of TNBC and benign breast epithelial cells. Furthermore, BCL2L14-ETV6 fusions prime partial epithelial-mesenchymal transition and endow resistance to paclitaxel treatment. Together, these data reveal AGRs as a class of underexplored genetic aberrations that could be pathological in breast cancer, and identify BCL2L14-ETV6 as a recurrent gene fusion in more aggressive form of TNBC tumors.


Assuntos
Rearranjo Gênico/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fusão Gênica/genética , Genômica/métodos , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Proteínas de Fusão Oncogênica/genética , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Sequenciamento Completo do Genoma
6.
Nat Genet ; 52(5): 505-515, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32251373

RESUMO

Active enhancers are frequently transcribed, yet the regulatory role of enhancer transcription remains debated. Here, we depleted the RNA polymerase II pausing and elongation factor Spt5 in activated mouse B cells and found that approximately 50% of enhancer-gene pairs showed co-regulated transcription, consistent with a potential functional requirement for enhancer transcription. In particular, Spt5 depletion led to loss of super-enhancer-promoter physical interaction and gene expression at the immunoglobulin heavy-chain locus (Igh), abrogating antibody class switch recombination. This defect correlated strictly with loss of enhancer transcription but did not affect acetylation of histone H3 at lysine 27, chromatin accessibility and occupancy of Mediator and cohesin at the enhancer. Strikingly, CRISPRa-mediated rescue of enhancer transcription in Spt5-depleted cells restored Igh gene expression. Our work suggests that Spt5-mediated enhancer transcription underlies the physical and functional interaction between a subset of active enhancers and their target promoters.


Assuntos
Elementos Facilitadores Genéticos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Transcrição Genética/genética , Acetilação , Animais , Proteínas de Ciclo Celular/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Expressão Gênica/genética , Rearranjo Gênico/genética , Switching de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
7.
PLoS Genet ; 16(3): e1008615, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32130223

RESUMO

The relative linear order of most genes on bacterial chromosomes is not conserved over evolutionary timescales. One explanation is that selection is weak, allowing recombination to randomize gene order by genetic drift. However, most chromosomal rearrangements are deleterious to fitness. In contrast, we propose the hypothesis that rearrangements in gene order are more likely the result of selection during niche adaptation (SNAP). Partial chromosomal duplications occur very frequently by recombination between direct repeat sequences. Duplicated regions may contain tens to hundreds of genes and segregate quickly unless maintained by selection. Bacteria exposed to non-lethal selections (for example, a requirement to grow on a poor nutrient) can adapt by maintaining a duplication that includes a gene that improves relative fitness. Further improvements in fitness result from the loss or inactivation of non-selected genes within each copy of the duplication. When genes that are essential in single copy are lost from different copies of the duplication, segregation is prevented even if the original selection is lifted. Functional gene loss continues until a new genetic equilibrium is reached. The outcome is a rearranged gene order. Mathematical modelling shows that this process of positive selection to adapt to a new niche can rapidly drive rearrangements in gene order to fixation. Signature features (duplication formation and divergence) of the SNAP model were identified in natural isolates from multiple species showing that the initial two steps in the SNAP process can occur with a remarkably high frequency. Further bioinformatic and experimental analyses are required to test if and to which extend the SNAP process acts on bacterial genomes.


Assuntos
Aclimatação/genética , Cromossomos Bacterianos/genética , Duplicação Gênica/genética , Rearranjo Gênico/genética , Seleção Genética/genética , Aberrações Cromossômicas , Evolução Molecular , Frequência do Gene/genética , Ordem dos Genes/genética , Genoma Bacteriano/genética , Modelos Teóricos , Filogenia
8.
Oncol Rep ; 43(3): 817-826, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32020234

RESUMO

As one of the most common types of renal cancer, clear cell renal cell carcinoma (ccRCC) in advanced stages constitutes a continued major challenge for uro­oncologists, as the identification of novel driver mutations and the development of novel targeted therapies against them remain an unmet need. Aberrations in anaplastic lymphoma kinase (ALK), a rational therapeutic target, as verified in lung cancer with ALK rearrangement, have been implicated in the pathogenesis of multiple human cancers. In the present study, we screened ALK expression in 87 pathologically defined ccRCCs via immunohistochemistry (IHC) using a newly developed rabbit anti­human ALK monoclonal antibody (clone D5F3). Four patients tested positive for ALK expression, as confirmed by IHC. Among them, 2 patients were further confirmed with fluorescence in situ hybridization (FISH) assay with the use of the Vysis LSI ALK dual color break­apart probe. Furthermore, we detected the existence of the echinoderm microtubule­associated protein­like 4/anaplastic lymphoma kinase (EML4­ALK) (E13:A20, variant 1) fusion gene in tumors from these two patients by using rapid amplification of cDNA ends (RACE)­coupled PCR sequencing and RT­PCR. Notably, we first showed that enforced EML4­ALK expression could significantly promote in vitro proliferation, clonogenic colony formation and apoptosis resistance in HK2 immortalized normal renal tubal epithelial cells and their in vivo outgrowth when injected into immunocompromised nude mice. Importantly, this pro­tumorigenic effect was completely abolished by the ALK­specific inhibitor crizotinib, indicating the potential effectiveness of ALK­specific inhibitors in treating ALK­rearranged ccRCC patients. Our data revealed that ALK fusions exist in adult ccRCC, providing a rationale for ALK inhibitor therapy in selected patients with ccRCC.


Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinogênese/genética , Carcinoma de Células Renais/genética , Proteínas de Fusão Oncogênica/genética , Adulto , Idoso , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Animais , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Feminino , Rearranjo Gênico/genética , Xenoenxertos , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/antagonistas & inibidores
9.
Sci Rep ; 10(1): 2077, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034268

RESUMO

Nuclear copies of the mitochondrial DNA (NUMTs) have already been described in several species. In this context, we identified and analysed 166 bovine NUMT regions with a total length of 430 kbp, representing about 0.02% of the cattle nuclear genome. Copies of all mitochondrial regions were detected in the nuclear genome, with distinct degrees of sequence similarity to the mitogenome. Some NUMT regions include large mitogenome segments and show high similarity to the organelle genome sequence. NUMT regions are frequently modified by insertion of repetitive sequences and by sequence rearrangements. We confirmed the existence of 29 NUMT regions by PCR amplification using DNA from the cow (Dominette) which was used to generate the bovine genome reference sequence, ruling out the possibility that these NUMTs could be artifacts of the genome assembly. As there are NUMT regions with high similarity to the mitogenome, special care is needed when designing primers for mitochondrial DNA amplification. Our results can therefore be used to avoid co-amplification of bovine nuclear sequences similar to mitochondrial DNA.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial/genética , Genoma/genética , Animais , Bovinos/genética , Rearranjo Gênico/genética , Genoma Mitocondrial/genética , Mitocôndrias/genética , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico/genética
10.
Nucleic Acids Res ; 48(5): 2544-2563, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32016395

RESUMO

The evolution of gene expression regulation has contributed to species differentiation. The 3' untranslated regions (3'UTRs) of mRNAs include regulatory elements that modulate gene expression; however, our knowledge of their implications in the divergence of bacterial species is currently limited. In this study, we performed genome-wide comparative analyses of mRNAs encoding orthologous proteins from the genus Staphylococcus and found that mRNA conservation was lost mostly downstream of the coding sequence (CDS), indicating the presence of high sequence diversity in the 3'UTRs of orthologous genes. Transcriptomic mapping of different staphylococcal species confirmed that 3'UTRs were also variable in length. We constructed chimeric mRNAs carrying the 3'UTR of orthologous genes and demonstrated that 3'UTR sequence variations affect protein production. This suggested that species-specific functional 3'UTRs might be specifically selected during evolution. 3'UTR variations may occur through different processes, including gene rearrangements, local nucleotide changes, and the transposition of insertion sequences. By extending the conservation analyses to specific 3'UTRs, as well as the entire set of Escherichia coli and Bacillus subtilis mRNAs, we showed that 3'UTR variability is widespread in bacteria. In summary, our work unveils an evolutionary bias within 3'UTRs that results in species-specific non-coding sequences that may contribute to bacterial diversity.


Assuntos
Regiões 3' não Traduzidas/genética , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Staphylococcus/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Elementos de DNA Transponíveis/genética , Rearranjo Gênico/genética , Genes Bacterianos , Hemólise , Nucleotídeos/genética , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ovinos , Especificidade da Espécie
11.
Nat Genet ; 52(3): 306-319, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32024998

RESUMO

About half of all cancers have somatic integrations of retrotransposons. Here, to characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 38 histological cancer subtypes within the framework of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. We identified 19,166 somatically acquired retrotransposition events, which affected 35% of samples and spanned a range of event types. Long interspersed nuclear element (LINE-1; L1 hereafter) insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, which sometimes leads to the removal of tumor-suppressor genes, and can induce complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage-fusion-bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications for the development of human tumors.


Assuntos
Carcinogênese/genética , Rearranjo Gênico/genética , Genoma Humano/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Neoplasias/genética , Retroelementos/genética , Humanos , Neoplasias/patologia
12.
Nat Genet ; 52(3): 294-305, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32024999

RESUMO

Chromatin is folded into successive layers to organize linear DNA. Genes within the same topologically associating domains (TADs) demonstrate similar expression and histone-modification profiles, and boundaries separating different domains have important roles in reinforcing the stability of these features. Indeed, domain disruptions in human cancers can lead to misregulation of gene expression. However, the frequency of domain disruptions in human cancers remains unclear. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumor types, we analyzed 288,457 somatic structural variations (SVs) to understand the distributions and effects of SVs across TADs. Notably, SVs can lead to the fusion of discrete TADs, and complex rearrangements markedly change chromatin folding maps in the cancer genomes. Notably, only 14% of the boundary deletions resulted in a change in expression in nearby genes of more than twofold.


Assuntos
Cromatina/genética , Rearranjo Gênico/genética , Genoma Humano/genética , Variação Estrutural do Genoma , Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Humanos
13.
Nature ; 578(7793): 112-121, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025012

RESUMO

A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes1-7. Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types8. Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions-as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2-7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and-in liver cancer-frequently activate the telomerase gene TERT. A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.


Assuntos
Variação Genética , Genoma Humano/genética , Neoplasias/genética , Rearranjo Gênico/genética , Genômica , Humanos , Mutagênese Insercional , Telomerase/genética
14.
BMC Evol Biol ; 20(1): 17, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005133

RESUMO

BACKGROUND: Drosophila subobscura exhibits a rich inversion polymorphism, with some adaptive inversions showing repeatable spatiotemporal patterns in frequencies related to temperature. Previous studies reported increased basal HSP70 protein levels in homokaryotypic strains for a warm-climate arrangement compared to a cold-climate one. These findings do not match the similar hsp70 genomic organization between arrangements, where gene expression levels are expected to be similar. In order to test this hypothesis and understand the molecular basis for hsp70 expression, we compared basal hsp70 mRNA levels in males and females, and analysed the 5' and 3' regulatory regions of hsp70 genes in warm- and cold-climate isochromosomal O3 + 4 + 7 and OST lines of D. subobscura. RESULTS: We observed comparable mRNA levels between the two arrangements and a sex-biased hsp70 gene expression. The number of heat-shock elements (HSEs) and GAGA sites on the promoters were identical amongst the OST and O3 + 4 + 7 lines analysed. This is also true for 3' AU-rich elements where most A and B copies of hsp70 have, respectively, two and one element in both arrangements. Beyond the regulatory elements, the only notable difference between both arrangements is the presence in 3' UTR of a 14 bp additional fragment after the stop codon in the hsp70A copy in five O3 + 4 + 7 lines, which was not found in any of the six OST lines. CONCLUSIONS: The equivalent hsp70 mRNA amounts in OST and O3 + 4 + 7 arrangements provide the first evidence of a parallelism between gene expression and genetic organization in D. subobscura lines having these arrangements. This is reinforced by the lack of important differential features in the number and structure of regulatory elements between both arrangements, despite the genetic differentiation observed when the complete 5' and 3' regulatory regions were considered. Therefore, the basal levels of hsp70 mRNA cannot account, in principle, for the adaptive variation of the two arrangements studied. Consequently, further studies are necessary to understand the intricate molecular mechanisms of hsp70 gene regulation in D. subobscura.


Assuntos
Adaptação Fisiológica/genética , Clima , Drosophila/genética , Regulação da Expressão Gênica , Rearranjo Gênico/genética , Genes de Insetos , Variação Genética , Proteínas de Choque Térmico HSP70/genética , Regiões 3' não Traduzidas/genética , Análise de Variância , Animais , Sequência de Bases , Sequência Conservada , Drosophila/fisiologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética
15.
Clin Chim Acta ; 505: 49-54, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32092317

RESUMO

BACKGROUND: Germline mutations in BRCA1 and BRCA2 (BRCA1/2) have been conventionally analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Nowadays, next-generation sequencing (NGS) is increasingly being used in clinical genetics. The aim of this study was to evaluate the performance of NGS BRCA1/2 assays by comparing them with the conventional method. MATERIALS AND METHODS: We did BRCA1/2 NGS assays of 108 breast and/or ovarian cancer patients whose BRCA1/2 mutation had been previously analyzed by Sanger sequencing and MLPA using TruSeq Custom Amplicon Design AFP2. Single-nucleotide variations (SNVs) and small insertions or deletions (InDels) were evaluated. In addition, we analyzed large genomic rearrangements (LGRs) using a coverage-based algorithm as well as a revised BRCA1/2 NGS assay (BRCAaccuTest PLUS), which additionally covered a BRCA1 promoter region. RESULTS: The NGS BRCA1/2 assay detected all 20 SNVs and 21 small InDels in 56 patients. Among seven LGRs detected by MLPA, six exonic LGRs were well identified by both NGS BRCA1/2 assays. One pathogenic LGR, located on a BRCA1 promoter region, was successfully identified using revised BRCAaccuTestPLUS. CONCLUSIONS: These results indicated that an NGS BRCA1/2 assay could detect most LGRs including BRCA1 promoter-region deletion as well as SNVs and small InDels. Therefore, it was applicable to clinical BRCA1/2 mutation tests.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Deleção de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regiões Promotoras Genéticas/genética , Adulto , Idoso , Algoritmos , Neoplasias da Mama/genética , Variações do Número de Cópias de DNA , Éxons , Feminino , Rearranjo Gênico/genética , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Mutação/genética , Neoplasias Ovarianas/genética , Polimorfismo de Nucleotídeo Único
16.
Genes (Basel) ; 11(1)2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936645

RESUMO

Repetitive DNA is a major organizational component of eukaryotic genomes, being intrinsically related with their architecture and evolution. Tandemly repeated satellite DNAs (satDNAs) can be found clustered in specific heterochromatin-rich chromosomal regions, building vital structures like functional centromeres and also dispersed within euchromatin. Interestingly, despite their association to critical chromosomal structures, satDNAs are widely variable among species due to their high turnover rates. This dynamic behavior has been associated with genome plasticity and chromosome rearrangements, leading to the reshaping of genomes. Here we present the current knowledge regarding satDNAs in the light of new genomic technologies, and the challenges in the study of these sequences. Furthermore, we discuss how these sequences, together with other repeats, influence genome architecture, impacting its evolution and association with disease.


Assuntos
Adaptação Fisiológica/genética , DNA Satélite/genética , DNA Satélite/metabolismo , Animais , Centrômero/genética , Centrômero/metabolismo , Cromossomos/genética , Elementos de DNA Transponíveis/genética , Eucariotos , Evolução Molecular , Rearranjo Gênico/genética , Genômica , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos
17.
Genes Chromosomes Cancer ; 59(2): 125-130, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31515871

RESUMO

Infant acute lymphoblastic leukemias (ALL) are rare hematological malignancies occurring in children younger than 1 year of age, most frequently associated with KMT2A rearrangements (KMT2A-r). The smaller subset without KMT2A-r, which represents 20% of infant ALL cases, is poorly characterized. Here we report two cases of chemotherapy-sensitive non-KMT2A-r infant ALL. Transcriptome analyses revealed identical ACIN1-NUTM1 gene fusions in both cases, derived from cryptic chromosomal rearrangements undetected by standard cytogenetic approaches. Two isoforms of the gene fusion, joining exons 3 or 4 of ACIN1 to exon 3 of NUTM1, were identified. Both fusion transcripts contained the functional DNA-binding SAP (SAF-A/B, Acinus, and PIAS) domain of ACIN1 and most of NUTM1. The detection of the ACIN1-NUTM1 fusion by RT-PCR allowed the molecular monitoring of minimal residual disease in a clinical setting. Based on publicly available genomic datasets and literature review, we predict that NUTM1 gene fusions are recurrent events in infant ALL. As such, we propose two clinically relevant assays to screen for NUTM1 rearrangements in bone marrow cells, independent of the fusion partner: NUMT1 immunohistochemistry and NUTM1 RNA expression. In sum, our study identifies ACIN1-NUTM1 as a recurrent and possibly cryptic fusion in non-KMT2A-r infant ALL, provides clinical tools to screen for NUTM1-rearranged leukemia and contributes to the refinement of this new subgroup.


Assuntos
Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Aberrações Cromossômicas , Citogenética , Fusão Gênica , Rearranjo Gênico/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imuno-Histoquímica , Recém-Nascido , Leucemia Mieloide Aguda/genética , Masculino , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo
18.
Theor Appl Genet ; 133(1): 187-199, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31587087

RESUMO

KEY MESSAGE: A novel tetraploid S. spontaneum with basic chromosome x = 10 was discovered, providing us insights in the origin and evolution in Saccharum species. Sugarcane (Saccharum spp., Poaceae) is a leading crop for sugar production providing 80% of the world's sugar. However, the genetic and genomic complexities of this crop such as its high polyploidy level and highly variable chromosome numbers have significantly hindered the studies in deciphering the genomic structure and evolution of sugarcane. Here, we developed the first set of oligonucleotide (oligo)-based probes based on the S. spontaneum genome (x = 8), which can be used to simultaneously distinguish each of the 64 chromosomes of octaploid S. spontaneum SES208 (2n = 8x = 64) through fluorescence in situ hybridization (FISH). By comparative FISH assay, we confirmed the chromosomal rearrangements of S. spontaneum (x = 8) and S. officinarum (2n = 8x = 80), the main contributors of modern sugarcane cultivars. In addition, we examined a S. spontaneum accession, Np-X, with 2n = 40 chromosomes, and we found that it was a tetraploid with the unusual basic chromosome number of x = 10. Assays at the cytological and DNA levels demonstrated its close relationship with S. spontaneum with basic chromosome number x = 8 (the most common accessions in S. spontaneum), confirming its S. spontaneum identity. Population genetic structure and phylogenetic relationship analyses between Np-X and 64 S. spontaneum accessions revealed that Np-X belongs to the ancient Pan-Malaysia group, indicating a close relationship to S. spontaneum with basic chromosome number of x = 8. This finding of a tetraploid S. spontaneum with basic chromosome number of x = 10 suggested a parallel evolution path of genomes and polyploid series in S. spontaneum with different basic chromosome numbers.


Assuntos
Cromossomos de Plantas/genética , Evolução Molecular , Genoma de Planta , Saccharum/genética , Ecótipo , Rearranjo Gênico/genética , Genética Populacional , Hibridização in Situ Fluorescente , Cariotipagem , Metáfase/genética , Filogenia , Análise de Sequência de DNA , Fatores de Tempo
19.
Gene ; 726: 144154, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31589962

RESUMO

In this work the complete chloroplast DNAs of Allium paradoxum and Allium ursinum, two edible species of Allium subg. Amerallium (the first lineage), were sequenced, assembled, annotated, and compared with complete Allium plastomes of the second and third evolutionary lines from GenBank database. The A. ursinum plastome contains 90 predicted genes (81 unique) including 5 pseudogenes, while A. paradoxum has 88 predicted genes (79 unique) including 19 pseudogenes. The comparative analysis has revealed that the A. paradoxum plastome differs markedly from those of other species. Due to many deletions, the A. paradoxum plastome is the shortest of known for Allium species, being only 145,819 bp long. The most prominent distinctions are (1) a 4825 bp long local inversion that spans from the ndhE to the rpl32 gene in the small single copy region and (2) pseudogenization, or the loss of all NADH-genes. In contrast, the plastome of A. ursinum - a species from the first evolutionary line (as well as A. paradoxum) - resembles the Allium species of the second and third evolutionary lines, showing no large rearrangements or discrepancies in gene content. It is unclear yet whether only A. paradoxum was affected by some evolutionary events or its close relatives from both sect. Briseis and other sections of Amerallium were altered as well. We speculate the sunlight-intolerant, shade-loving nature of A. paradoxum and the impairment of the ndh genes in its plastome could be interrelated phenomena.


Assuntos
Allium/genética , Rearranjo Gênico/genética , Genes de Plantas/genética , Cebolas/genética , DNA de Cloroplastos/genética , DNA de Plantas/genética , Evolução Molecular , Genoma de Cloroplastos/genética , Genoma de Planta/genética , Filogenia , Folhas de Planta/genética , Pseudogenes/genética , Análise de Sequência de DNA/métodos
20.
Acta Cytol ; 64(4): 378-385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31661685

RESUMO

BACKGROUND: A case of peritoneal mesothelioma with an anaplastic lymphoma kinase (ALK) translocation was identified, and we conducted further studies to obtain diagnostic and therapeutic insights. We believe that this is the first report describing the cytology of this new tumor type. CASE: A teenage woman was referred for severe pleural effusion. Enhanced computed tomography indicated an abdominal mass with ascites. Laparoscopy revealed tumor dissemination from the pelvis to the upper abdomen. Because a high-grade serous carcinoma was suspected, ascitic cytology and biopsy were performed. Cytologically, the tumor displayed characteristics of both adenocarcinoma and reactive or neoplastic mesothelial cells. After extensive pathological evaluation, the tumor was diagnosed as malignant peritoneal mesothelioma. To verify the diagnosis and aid in developing a therapeutic strategy, several companion diagnostics were tried. Surprisingly, the tumor was ALK-positive, and ALK recombination was confirmed by an ALK break-apart test. Retrospectively, cells and tissue specimens were stained with ALK intercalated antibody-enhanced polymer. Tumor cells were clearly distinguished from the nonneoplastic background. Recombination in ALK was reconfirmed by the National Cancer Center Japan, and the patient was enrolled in a clinical trial for alectinib. CONCLUSION: Companion diagnostics-based cytology may provide a useful means of monitoring and evaluating a molecular-targeted therapy.


Assuntos
Quinase do Linfoma Anaplásico/genética , Ascite/diagnóstico , Ascite/genética , Rearranjo Gênico/genética , Mesotelioma/diagnóstico , Mesotelioma/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adolescente , Citodiagnóstico/métodos , Feminino , Humanos , Estudos Retrospectivos
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