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1.
BMC Evol Biol ; 19(1): 124, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31215393

RESUMO

BACKGROUND: Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members. RESULTS: Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete MmucT (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNAIleTAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members. CONCLUSIONS: Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.


Assuntos
Genoma Bacteriano , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/genética , RNA Bacteriano/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Aminoacil-tRNA Sintetases/genética , Bacteriófagos/genética , Tamanho do Genoma , Genômica , Anotação de Sequência Molecular , Mycobacterium/classificação , Filogenia , Plasmídeos/genética , RNA não Traduzido/química , Ribonuclease P/genética , Inversão de Sequência
2.
Nat Genet ; 51(6): 1024-1034, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31133748

RESUMO

The mouse X-inactivation center (Xic) locus represents a powerful model for understanding the links between genome architecture and gene regulation, with the non-coding genes Xist and Tsix showing opposite developmental expression patterns while being organized as an overlapping sense/antisense unit. The Xic is organized into two topologically associating domains (TADs) but the role of this architecture in orchestrating cis-regulatory information remains elusive. To explore this, we generated genomic inversions that swap the Xist/Tsix transcriptional unit and place their promoters in each other's TAD. We found that this led to a switch in their expression dynamics: Xist became precociously and ectopically upregulated, both in male and female pluripotent cells, while Tsix expression aberrantly persisted during differentiation. The topological partitioning of the Xic is thus critical to ensure proper developmental timing of X inactivation. Our study illustrates how the genomic architecture of cis-regulatory landscapes can affect the regulation of mammalian developmental processes.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , RNA Longo não Codificante/genética , Inativação do Cromossomo X , Animais , Diferenciação Celular/genética , Expressão Ectópica do Gene , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Inativação Gênica , Loci Gênicos , Masculino , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Inversão de Sequência , Transcrição Genética
3.
mSphere ; 4(2)2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971446

RESUMO

Most urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC), which depends on an extracellular organelle (type 1 pili) for adherence to bladder cells during infection. Type 1 pilus expression is partially regulated by inversion of a piece of DNA referred to as fimS, which contains the promoter for the fim operon encoding type 1 pili. fimS inversion is regulated by up to five recombinases collectively known as Fim recombinases. These Fim recombinases are currently known to regulate two other switches: the ipuS and hyxS switches. A long-standing question has been whether the Fim recombinases regulate the inversion of other switches, perhaps to coordinate expression for adhesion or virulence. We answered this question using whole-genome sequencing with a newly developed algorithm (structural variation detection using relative entropy [SVRE]) for calling structural variations using paired-end short-read sequencing. SVRE identified all of the previously known switches, refining the specificity of which recombinases act at which switches. Strikingly, we found no new inversions that were mediated by the Fim recombinases. We conclude that the Fim recombinases are each highly specific for a small number of switches. We hypothesize that the unlinked Fim recombinases have been recruited to regulate fimS, and fimS only, as a secondary locus; this further implies that regulation of type 1 pilus expression (and its role in gastrointestinal and/or genitourinary colonization) is important enough, on its own, to influence the evolution and maintenance of multiple additional genes within the accessory genome of E. coli IMPORTANCE UTI is a common ailment that affects more than half of all women during their lifetime. The leading cause of UTIs is UPEC, which relies on type 1 pili to colonize and persist within the bladder during infection. The regulation of type 1 pili is remarkable for an epigenetic mechanism in which a section of DNA containing a promoter is inverted. The inversion mechanism relies on what are thought to be dedicated recombinase genes; however, the full repertoire for these recombinases is not known. We show here that there are no additional targets beyond those already identified for the recombinases in the entire genome of two UPEC strains, arguing that type 1 pilus expression itself is the driving evolutionary force for the presence of these recombinase genes. This further suggests that targeting the type 1 pilus is a rational alternative nonantibiotic strategy for the treatment of UTI.


Assuntos
Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Inversão de Sequência , Escherichia coli Uropatogênica/genética , Algoritmos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Entropia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/química , Humanos , Integrases/química , Integrases/genética , Regiões Promotoras Genéticas , Infecções Urinárias/microbiologia , Virulência/genética
4.
Genome ; 62(5): 317-328, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30998854

RESUMO

Picea asperata and P. crassifolia have sympatric ranges and are closely related, but the differences between these species at the plastome level are unknown. To better understand the patterns of variation among Picea plastomes, the complete plastomes of P. asperata and P. crassifolia were sequenced. Then, the plastomes were compared with the complete plastomes of P. abies and P. morrisonicola, which are closely and distantly related to the focal species, respectively. We also used these sequences to construct phylogenetic trees to determine the relationships among and between the four species as well as additional taxa from Pinaceae and other gymnosperms. Analysis of our sequencing data allowed us to identify 438 single nucleotide polymorphism (SNPs) point mutation events, 95 indel events, four inversion events, and seven highly variable regions, including six gene spacer regions (psbJ-petA, trnT-psaM, trnS-trnD, trnL-rps4, psaC-ccsA, and rps7-trnL) and one gene (ycf1). The highly variable regions are appropriate targets for future use in the phylogenetic reconstructions of closely related, sympatric species of Picea as well as Pinaceae in general.


Assuntos
Genomas de Plastídeos/genética , Picea/genética , Simpatria , Mutação INDEL , Filogenia , Plastídeos/genética , Polimorfismo de Nucleotídeo Único/genética , Inversão de Sequência/genética
6.
Genome Res ; 28(6): 910-920, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29776991

RESUMO

For many years, inversions have been proposed to be a direct driving force in speciation since they suppress recombination when heterozygous. Inversions are the most common large-scale differences among humans and great apes. Nevertheless, they represent large events easily distinguishable by classical cytogenetics, whose resolution, however, is limited. Here, we performed a genome-wide comparison between human, great ape, and macaque genomes using the net alignments for the most recent releases of genome assemblies. We identified a total of 156 putative inversions, between 103 kb and 91 Mb, corresponding to 136 human loci. Combining literature, sequence, and experimental analyses, we analyzed 109 of these loci and found 67 regions inverted in one or multiple primates, including 28 newly identified inversions. These events overlap with 81 human genes at their breakpoints, and seven correspond to sites of recurrent rearrangements associated with human disease. This work doubles the number of validated primate inversions larger than 100 kb, beyond what was previously documented. We identified 74 sites of errors, where the sequence has been assembled in the wrong orientation, in the reference genomes analyzed. Our data serve two purposes: First, we generated a map of evolutionary inversions in these genomes representing a resource for interrogating differences among these species at a functional level; second, we provide a list of misassembled regions in these primate genomes, involving over 300 Mb of DNA and 1978 human genes. Accurately annotating these regions in the genome references has immediate applications for evolutionary and biomedical studies on primates.


Assuntos
Inversão Cromossômica/genética , Genoma Humano/genética , Primatas/genética , Inversão de Sequência/genética , Animais , Evolução Molecular , Humanos , Anotação de Sequência Molecular , Pan troglodytes/genética
7.
Genetics ; 209(2): 425-438, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29610215

RESUMO

In many organisms, telomeric sequences can be located internally on the chromosome in addition to their usual positions at the ends of the chromosome. In humans, such interstitial telomeric sequences (ITSs) are nonrandomly associated with translocation breakpoints in tumor cells and with chromosome fragile sites (regions of the chromosome that break in response to perturbed DNA replication). We previously showed that ITSs in yeast generated several different types of instability, including terminal inversions (recombination between the ITS and the "true" chromosome telomere) and point mutations in DNA sequences adjacent to the ITS. In the current study, we examine the genetic control of these events. We show that the terminal inversions occur by the single-strand annealing pathway of DNA repair following the formation of a double-stranded DNA break within the ITS. The point mutations induced by the ITS require the error-prone DNA polymerase ζ. Unlike the terminal inversions, these events are not initiated by a double-stranded DNA break, but likely result from the error-prone repair of a single-stranded DNA gap or recruitment of DNA polymerase ζ in the absence of DNA damage.


Assuntos
Instabilidade Genômica , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Telômero/genética , Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Genoma Fúngico , Inversão de Sequência
8.
J Proteome Res ; 17(3): 1258-1268, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29336158

RESUMO

The spatial distribution of genes in chromosomes seems not to be random. For instance, only 10% of genes are transcribed from bidirectional promoters in humans, and many more are organized into larger clusters. This raises intriguing questions previously asked by different authors. We would like to add a few more questions in this context, related to gene orientation inversions. Does gene orientation (inversion) follow a random pattern? Is it relevant to biological activity somehow? We define a new kind of network coined as the gene orientation inversion network (GOIN). GOIN's complex network encodes short- and long-range patterns of inversion of the orientation of pairs of gene in the chromosome. We selected Plasmodium falciparum as a case of study due to the high relevance of this parasite to public health (causal agent of malaria). We constructed here for the first time all of the GOINs for the genome of this parasite. These networks have an average of 383 nodes (genes in one chromosome) and 1314 links (pairs of gene with inverse orientation). We calculated node centralities and other parameters of these networks. These numerical parameters were used to study different properties of gene inversion patterns, for example, distribution, local communities, similarity to Erdös-Rényi random networks, randomness, and so on. We find clues that seem to indicate that gene orientation inversion does not follow a random pattern. We noted that some gene communities in the GOINs tend to group genes encoding for RIFIN-related proteins in the proteome of the parasite. RIFIN-like proteins are a second family of clonally variant proteins expressed on the surface of red cells infected with Plasmodium falciparum. Consequently, we used these centralities as input of machine learning (ML) models to predict the RIFIN-like activity of 5365 proteins in the proteome of Plasmodium sp. The best linear ML model found discriminates RIFIN-like from other proteins with sensitivity and specificity 70-80% in training and external validation series. All of these results may point to a possible biological relevance of gene orientation inversion not directly dependent on genetic sequence information. This work opens the gate to the use of GOINs as a tool for the study of the structure of chromosomes and the study of protein function in proteome research.


Assuntos
Cromossomos/química , Redes Reguladoras de Genes , Genes de Protozoários , Proteínas de Membrana/genética , Plasmodium falciparum/genética , Proteoma/genética , Proteínas de Protozoários/genética , Inversão de Sequência , Eritrócitos/parasitologia , Regulação da Expressão Gênica , Humanos , Aprendizado de Máquina , Proteínas de Membrana/metabolismo , Família Multigênica , Plasmodium falciparum/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Software
9.
Gene ; 645: 113-118, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29273555

RESUMO

Genomic rearrangements, such as intragenic deletions and duplications, are the most prevalent types of mutation in the DMD gene, and DMD mutations underlie Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Using multiplex ligation dependent probe amplification (MLPA) and DMD gene-targeted sequencing, we performed a molecular characterization of two cases of complex noncontiguous duplication rearrangements that involved inverted duplications. The breakpoint sequences were analyzed to investigate the mechanisms of the rearrangement. The two cases shared the same duplication events (Dup-nml-Dup/inv), and both involved microhomology and small insertions at the breakpoints. Additionally, in case 1, SNP sequencing results indicated that the de novo duplication mutation arose in the allele that originated from the grandfather. This study has identified a novel type of DMD complex rearrangement and provides insight into the molecular basis of this genomic rearrangement.


Assuntos
Duplicação Cromossômica , Deficiências do Desenvolvimento/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Criança , Pontos de Quebra do Cromossomo , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Análise de Sequência de DNA , Inversão de Sequência
10.
Genetica ; 146(1): 101-113, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29170851

RESUMO

The plant chloroplast (cp) genome is a highly conserved structure which is beneficial for evolution and systematic research. Currently, numerous complete cp genome sequences have been reported due to high throughput sequencing technology. However, there is no complete chloroplast genome of genus Dodonaea that has been reported before. To better understand the molecular basis of Dodonaea viscosa chloroplast, we used Illumina sequencing technology to sequence its complete genome. The whole length of the cp genome is 159,375 base pairs (bp), with a pair of inverted repeats (IRs) of 27,099 bp separated by a large single copy (LSC) 87,204 bp, and small single copy (SSC) 17,972 bp. The annotation analysis revealed a total of 115 unique genes of which 81 were protein coding, 30 tRNA, and four ribosomal RNA genes. Comparative genome analysis with other closely related Sapindaceae members showed conserved gene order in the inverted and single copy regions. Phylogenetic analysis clustered D. viscosa with other species of Sapindaceae with strong bootstrap support. Finally, a total of 249 SSRs were detected. Moreover, a comparison of the synonymous (Ks) and nonsynonymous (Ka) substitution rates in D. viscosa showed very low values. The availability of cp genome reported here provides a valuable genetic resource for comprehensive further studies in genetic variation, taxonomy and phylogenetic evolution of Sapindaceae family. In addition, SSR markers detected will be used in further phylogeographic and population structure studies of the species in this genus.


Assuntos
Genoma de Cloroplastos , Sapindaceae/genética , Genômica , Repetições de Microssatélites , Filogenia , Sapindaceae/classificação , Inversão de Sequência
11.
Genome Med ; 9(1): 90, 2017 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-29070057

RESUMO

BACKGROUND: Massively parallel DNA sequencing, such as exome sequencing, has become a routine clinical procedure to identify pathogenic variants responsible for a patient's phenotype. Exome sequencing has the capability of reliably identifying inherited and de novo single-nucleotide variants, small insertions, and deletions. However, due to the use of 100-300-bp fragment reads, this platform is not well powered to sensitively identify moderate to large structural variants (SV), such as insertions, deletions, inversions, and translocations. METHODS: To overcome these limitations, we used next-generation mapping (NGM) to image high molecular weight double-stranded DNA molecules (megabase size) with fluorescent tags in nanochannel arrays for de novo genome assembly. We investigated the capacity of this NGM platform to identify pathogenic SV in a series of patients diagnosed with Duchenne muscular dystrophy (DMD), due to large deletions, insertion, and inversion involving the DMD gene. RESULTS: We identified deletion, duplication, and inversion breakpoints within DMD. The sizes of deletions were in the range of 45-250 Kbp, whereas the one identified insertion was approximately 13 Kbp in size. This method refined the location of the break points within introns for cases with deletions compared to current polymerase chain reaction (PCR)-based clinical techniques. Heterozygous SV were detected in the known carrier mothers of the DMD patients, demonstrating the ability of the method to ascertain carrier status for large SV. The method was also able to identify a 5.1-Mbp inversion involving the DMD gene, previously identified by RNA sequencing. CONCLUSIONS: We showed the ability of NGM technology to detect pathogenic structural variants otherwise missed by PCR-based techniques or chromosomal microarrays. NGM is poised to become a new tool in the clinical genetic diagnostic strategy and research due to its ability to sensitively identify large genomic variations.


Assuntos
Mapeamento Cromossômico , Genoma Humano , Variação Estrutural do Genoma , Distrofia Muscular de Duchenne/genética , Estudos de Coortes , Análise Mutacional de DNA , Distrofina/genética , Feminino , Deleção de Genes , Triagem de Portadores Genéticos , Humanos , Masculino , Mães , Distrofia Muscular de Duchenne/diagnóstico , Mutagênese Insercional , Inversão de Sequência
13.
Mol Microbiol ; 106(3): 409-418, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28833671

RESUMO

We recently proposed a model for targeted, conservative cointegrate formation between DNA molecules each containing a copy of IS26, that involves Tnp26-catalyzed strand exchange occurring at either the two left ends or the two right ends of the IS. Here, this model was validated by altering the bases at the outer left terminus, right terminus or both termini of one IS26. The correct bases at both ends were required in the untargeted replicative mode. However, when only one end was altered in one participating IS the frequency of targeted, conservative cointegrate formation was not reduced. The distribution of the altered bases in the cointegrates confirmed that the reaction occurred at the end where the terminal bases of both IS were correct, and cointegrates were not formed when both ends of the same IS were altered. The terminal bases of the active IS26 were also required to support deletion of the aphA1a translocatable unit (TU) from Tn4352B. The choices made by an incoming TU with a wild-type IS26 when the target plasmid included one wild-type IS26 and one with a frameshift in tnp26 demonstrated that Tnp26 exhibits a strong preference for cis action.


Assuntos
Elementos de DNA Transponíveis/genética , Mutagênese Insercional/genética , Sequência de Bases/genética , Replicação do DNA , Escherichia coli/genética , Modelos Moleculares , Plasmídeos/genética , Inversão de Sequência/genética
14.
Virology ; 511: 330-343, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28803676

RESUMO

Ranaviruses are emerging pathogens of fish, amphibians, and reptiles that threaten aquatic animal industries and wildlife worldwide. Our objective was to genetically characterize ranaviruses isolated during separate bullfrog Lithobates catesbeianus die-offs that occurred eight years apart on the same North American farm. The earlier outbreak was due to a highly pathogenic strain of common midwife toad virus (CMTV) previously known only from Europe and China. The later outbreak was due to a chimeric ranavirus that displayed a novel genome arrangement and a DNA backbone typical for Frog virus 3 (FV3) strains except for interspersed fragments acquired through recombination with the CMTV isolated earlier. Both bullfrog ranaviruses are more pathogenic than wild-type FV3 suggesting recombination may have resulted in the increased pathogenicity observed in the ranavirus isolated in the later outbreak. Our study underscores the role international trade in farmed bullfrogs may have played in the global dissemination of highly pathogenic ranaviruses.


Assuntos
Infecções por Vírus de DNA/veterinária , Variação Genética , Ranavirus/classificação , Ranavirus/genética , Recombinação Genética , Inversão de Sequência , Animais , Infecções por Vírus de DNA/virologia , DNA Viral/química , DNA Viral/genética , América do Norte , Rana catesbeiana/virologia , Análise de Sequência de DNA
15.
Blood ; 130(3): 340-347, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28490568

RESUMO

Direct detection of F8 and F9 sequence variants in maternal plasma of hemophilia carriers has been demonstrated by microfluidics digital PCR. Noninvasive prenatal assessment of the most clinically relevant group of sequence variants among patients with hemophilia, namely, those involving int22h-related inversions disrupting the F8 gene, poses additional challenges because of its molecular complexity. We investigated the use of droplet digital PCR (ddPCR) and targeted massively parallel sequencing (MPS) for maternal plasma DNA analysis to noninvasively determine fetal mutational status in pregnancies at risk for hemophilia. We designed family-specific ddPCR assays to detect causative sequence variants scattered across the F8 and F9 genes. A haplotype-based approach coupled with targeted MPS was applied to deduce fetal genotype by capturing a 7.6-Mb region spanning the F8 gene in carriers with int22h-related inversions. The ddPCR analysis correctly determined fetal hemophilia status in 15 at-risk pregnancies in samples obtained from 8 to 42 weeks of gestation. There were 3 unclassified samples, but no misclassification. Detailed fetal haplotype maps of the F8 gene region involving int22h-related inversions obtained through targeted MPS enabled correct diagnoses of fetal mutational status in 3 hemophilia families. Our data suggest it is feasible to apply targeted MPS to interrogate maternally inherited F8 int22h-related inversions, whereas ddPCR represents an affordable approach for the identification of F8 and F9 sequence variants in maternal plasma. These advancements may bring benefits for the pregnancy management for carriers of hemophilia sequence variants; in particular, the common F8 int22h-related inversions, associated with the most severe clinical phenotype.


Assuntos
Fator VIII/genética , Doenças Fetais/diagnóstico , Hemofilia A/diagnóstico , Heterozigoto , Diagnóstico Pré-Natal/métodos , Inversão de Sequência , Adulto , Fator IX/genética , Fator IX/metabolismo , Fator VIII/metabolismo , Feminino , Doenças Fetais/sangue , Doenças Fetais/genética , Doenças Fetais/patologia , Feto , Idade Gestacional , Hemofilia A/sangue , Hemofilia A/genética , Hemofilia A/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dispositivos Lab-On-A-Chip , Masculino , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Gravidez , Diagnóstico Pré-Natal/instrumentação
16.
Thromb Haemost ; 117(8): 1478-1485, 2017 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-28492696

RESUMO

Haemophilia A (HA) is a common X-linked recessive bleeding disorder and almost one half of patients with severe HA are caused by intron 22 inversion (Inv22) in the F8. Inv22 is considered to be almost exclusively of meiotic origin in germ cells during spermatogenesis and only one mosaic Inv22 female carrier with the mutation possibly occurring during mitosis of the embryo has been reported so far. Previously we have identified a novel complex recombination mediated by int22h copies in a sporadic severe HA pedigree and herein we have localised the sequences flanking the breakpoint region using genome walking technique, AccuCopy technique, gene chip and real-time PCR. The disease causing genetic variant registered an 18.1 kb deletion including part of int22h-1 through the intron 23 of F8 and a 113.3 kb duplication of part of int22h-2 through the intron 1 of TMLHE inserted in the religated region of the F8. Two intrinsically linked mechanisms of recombination-dependent DNA replication: microhomology-mediated break-induced replication (MMBIR) followed by break-induced replication (BIR) might be responsible for the incident of the complex recombination during early embryogenesis of the proband's mother.


Assuntos
Fator VIII/genética , Deleção de Genes , Duplicação Gênica , Hemofilia A/genética , Íntrons , Oxigenases de Função Mista/genética , Recombinação Genética , Inversão de Sequência , Passeio de Cromossomo , Cromossomos Humanos X , Análise Mutacional de DNA , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Predisposição Genética para Doença , Hemofilia A/sangue , Hemofilia A/diagnóstico , Hemofilia A/embriologia , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Mosaicismo , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Índice de Gravidade de Doença
17.
Sci Rep ; 7(1): 928, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28424528

RESUMO

A multiple DNA inversion system, the shufflon, exists in incompatibility (Inc) I1 and I2 plasmids. The shufflon generates variants of the PilV protein, a minor component of the thin pilus. The shufflon is one of the most difficult regions for de novo genome assembly because of its structural diversity even in an isolated bacterial clone. We determined complete genome sequences, including those of IncI2 plasmids carrying mcr-1, of three Escherichia coli strains using single-molecule, real-time (SMRT) sequencing and Illumina sequencing. The sequences assembled using only SMRT sequencing contained misassembled regions in the shufflon. A hybrid analysis using SMRT and Illumina sequencing resolved the misassembled region and revealed that the three IncI2 plasmids, excluding the shufflon region, were highly conserved. Moreover, the abundance ratio of whole-shufflon structures could be determined by quantitative structural variation analysis of the SMRT data, suggesting that a remarkable heterogeneity of whole-shufflon structural variations exists in IncI2 plasmids. These findings indicate that remarkable rearrangement regions should be validated using both long-read and short-read sequencing data and that the structural variation of PilV in the shufflon might be closely related to phenotypic heterogeneity of plasmid-mediated transconjugation involved in horizontal gene transfer even in bacterial clonal populations.


Assuntos
DNA Bacteriano/química , Escherichia coli/genética , Plasmídeos/genética , Inversão de Sequência , DNA Bacteriano/genética , Escherichia coli/química , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Genéticos , Plasmídeos/química , Análise de Sequência de DNA
18.
BMC Genomics ; 18(1): 268, 2017 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-28356070

RESUMO

BACKGROUND: Genome rearrangement describes gross changes of chromosomal regions, plays an important role in evolutionary biology and has profound impacts on phenotype in organisms ranging from microbes to humans. With more and more complete genomes accomplished, lots of genomic comparisons have been conducted in order to find genome rearrangements and the mechanisms which underlie the rearrangement events. In our opinion, genomic comparison of different individuals/strains within the same species (pan-genome) is more helpful to reveal the mechanisms for genome rearrangements since genomes of the same species are much closer to each other. RESULTS: We study the mechanism for inversion events via core-genome scaffold comparison of different strains within the same species. We focus on two kinds of bacteria, Pseudomonas aeruginosa and Escherichia coli, and investigate the inversion events among different strains of the same species. We find an interesting phenomenon that long (larger than 10,000 bp) inversion regions are flanked by a pair of Inverted Repeats (IRs). This mechanism can also explain why the breakpoint reuses for inversion events happen. We study the prevalence of the phenomenon and find that it is a major mechanism for inversions. The other observation is that for different rearrangement events such as transposition and inverted block interchange, the two ends of the swapped regions are also associated with repeats so that after the rearrangement operations the two ends of the swapped regions remain unchanged. To our knowledge, this is the first time such a phenomenon is reported for transposition event. CONCLUSIONS: In both Pseudomonas aeruginosa and Escherichia coli strains, IRs were found at the two ends of long sequence inversions. The two ends of the inversion remained unchanged before and after the inversion event. The existence of IRs can explain the breakpoint reuse phenomenon. We also observed that other rearrangement operations such as transposition, inverted transposition, and inverted block interchange, had repeats (not necessarily inverted) at the ends of each segment, where the ends remained unchanged before and after the rearrangement operations. This suggests that the conservation of ends could possibly be a popular phenomenon in many types of chromosome rearrangement events.


Assuntos
Escherichia coli/genética , Genoma Bacteriano , Pseudomonas aeruginosa/genética , Inversão de Sequência/genética , Hibridização Genômica Comparativa , Rearranjo Gênico
19.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 1): 51-53, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28045394

RESUMO

Earlier studies have found that the occurrence of inverse sequence identity in proteins is not indicative of three-dimensional similarity, but rather leads to different folds or unfolded proteins. Short helices, however, frequently keep their conformations when their sequences are inverted. To explore the impact of sequence inversion on long helices, revRM6, with the inverse amino-acid sequence relative to RM6, a highly stable variant of the ColE1 Rop protein, was engineered. RM6 is a highly regular four-α-helical bundle that serves as a model system for protein-folding studies. Here, the crystallization and preliminary crystallographic characterization of revRM6 are reported. The protein was overexpressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belonged to space group P41212, with unit-cell parameters a = b = 44.98, c = 159.74 Å, and diffracted to a resolution of 3.45 Å.


Assuntos
Proteínas de Bactérias/química , Escherichia coli/genética , Engenharia de Proteínas , Proteínas de Ligação a RNA/química , Inversão de Sequência , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli/metabolismo , Expressão Gênica , Plasmídeos/química , Plasmídeos/metabolismo , Conformação Proteica em alfa-Hélice , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difração de Raios X
20.
BMC Genomics ; 18(1): 65, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28073353

RESUMO

BACKGROUND: Although many algorithms are now available that aim to characterize different classes of structural variation, discovery of balanced rearrangements such as inversions remains an open problem. This is mainly due to the fact that breakpoints of such events typically lie within segmental duplications or common repeats, which reduces the mappability of short reads. The algorithms developed within the 1000 Genomes Project to identify inversions are limited to relatively short inversions, and there are currently no available algorithms to discover large inversions using high throughput sequencing technologies. RESULTS: Here we propose a novel algorithm, VALOR, to discover large inversions using new sequencing methods that provide long range information such as 10X Genomics linked-read sequencing, pooled clone sequencing, or other similar technologies that we commonly refer to as long range sequencing. We demonstrate the utility of VALOR using both pooled clone sequencing and 10X Genomics linked-read sequencing generated from the genome of an individual from the HapMap project (NA12878). We also provide a comprehensive comparison of VALOR against several state-of-the-art structural variation discovery algorithms that use whole genome shotgun sequencing data. CONCLUSIONS: In this paper, we show that VALOR is able to accurately discover all previously identified and experimentally validated large inversions in the same genome with a low false discovery rate. Using VALOR, we also predicted a novel inversion, which we validated using fluorescent in situ hybridization. VALOR is available at https://github.com/BilkentCompGen/VALOR.


Assuntos
Genômica/métodos , Inversão de Sequência/genética , Algoritmos , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequenciamento Completo do Genoma
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