RESUMO
The RNA-guided Cas9 system is a versatile tool for genome editing. Here, we established a RNA-guided endonuclease (RGEN) system as an in vivo desired-target mutator (DTM) in maize to reduce the linkage drag during breeding procedure, using the LIGULELESS1 (LG1) locus as a proof-of-concept. Our system showed 51.5%-91.2% mutation frequency in T0 transgenic plants. We then crossed the T1 plants stably expressing DTM with six diverse recipient maize lines and found that 11.79%-28.71% of the plants tested were mutants induced by the DTM effect. Analysis of successive F2 plants indicated that the mutations induced by the DTM effect were largely heritable. Moreover, DTM-generated hybrids had significantly smaller leaf angles that were reduced more than 50% when compared with that of the wild type. Planting experiments showed that DTM-generated maize plants can be grown with significantly higher density and hence greater yield potential. Our work demonstrate that stably expressed RGEN could be implemented as an in vivoDTM to rapidly generate and spread desired mutations in maize through hybridization and subsequent backcrossing, and hence bypassing the linkage drag effect in convention introgression methodology. This proof-of-concept experiment can be a potentially much more efficient breeding strategy in crops employing the RNA-guided Cas9 genome editing.
Assuntos
Mutação , Melhoramento Vegetal/métodos , RNA Guia de Cinetoplastídeos , Zea mays/genética , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR , Endonucleases/genética , Edição de Genes , Padrões de Herança , Taxa de Mutação , Plantas Geneticamente Modificadas/genética , Análise de Sequência de DNARESUMO
The aim of the present study was to assess possible adverse effects of transgene expression in leaves of field-grown barley relative to the influence of genetic background and the effect of plant interaction with arbuscular mycorrhizal fungi. We conducted transcript profiling, metabolome profiling, and metabolic fingerprinting of wild-type accessions and barley transgenics with seed-specific expression of (1,3-1, 4)-beta-glucanase (GluB) in Baronesse (B) as well as of transgenics in Golden Promise (GP) background with ubiquitous expression of codon-optimized Trichoderma harzianum endochitinase (ChGP). We found more than 1,600 differential transcripts between varieties GP and B, with defense genes being strongly overrepresented in B, indicating a divergent response to subclinical pathogen challenge in the field. In contrast, no statistically significant differences between ChGP and GP could be detected based on transcriptome or metabolome analysis, although 22 genes and 4 metabolites were differentially abundant when comparing GluB and B, leading to the distinction of these two genotypes in principle component analysis. The coregulation of most of these genes in GluB and GP, as well as simple sequence repeat-marker analysis, suggests that the distinctive alleles in GluB are inherited from GP. Thus, the effect of the two investigated transgenes on the global transcript profile is substantially lower than the effect of a minor number of alleles that differ as a consequence of crop breeding. Exposing roots to the spores of the mycorrhizal Glomus sp. had little effect on the leaf transcriptome, but central leaf metabolism was consistently altered in all genotypes.
Assuntos
Regulação da Expressão Gênica de Plantas , Hordeum/genética , Hordeum/metabolismo , Perfilação da Expressão Gênica , Glucana 1,3-beta-Glucosidase/genética , Glucana 1,3-beta-Glucosidase/metabolismo , Metaboloma , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente ModificadasRESUMO
In Alzheimer's disease (AD), early deficits in learning and memory are a consequence of synaptic modification induced by toxic beta-amyloid oligomers (oAbeta). To identify immediate molecular targets downstream of oAbeta binding, we prepared synaptoneurosomes from prefrontal cortex of control and incipient AD (IAD) patients, and isolated mRNAs for comparison of gene expression. This novel approach concentrates synaptic mRNA, thereby increasing the ratio of synaptic to somal mRNA and allowing discrimination of expression changes in synaptically localized genes. In IAD patients, global measures of cognition declined with increasing levels of dimeric Abeta (dAbeta). These patients also showed increased expression of neuroplasticity related genes, many encoding 3'UTR consensus sequences that regulate translation in the synapse. An increase in mRNA encoding the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) was paralleled by elevated expression of the corresponding protein in IAD. These results imply a functional impact on synaptic transmission as GluR2, if inserted, maintains the receptors in a low conductance state. Some overexpressed genes may induce early deficits in cognition and others compensatory mechanisms, providing targets for intervention to moderate the response to dAbeta.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Perfilação da Expressão Gênica , Plasticidade Neuronal/genética , Neurônios , Sinaptossomos/fisiologia , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Análise por Conglomerados , Cognição/fisiologia , Progressão da Doença , Humanos , Análise em Microsséries , Neurônios/fisiologia , Neurônios/ultraestrutura , Testes Neuropsicológicos , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiologia , RNA Mensageiro/análise , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Sinaptossomos/ultraestruturaRESUMO
BACKGROUND: The amphotropic murine leukemia viruses (MuLV-A's) are naturally occurring, exogenously acquired gammaretroviruses that are indigenous to the Southern California wild mice. These viruses replicate in a wide range of cell types including human cells in vitro and they can cause both hematological and neurological disorders in feral as well as in the inbred laboratory mice. Since MuLV-A's also exhibit discrete interference and neutralization properties, the envelope proteins of these viruses have been extremely useful for studying virus-host cell interactions and as vehicles for transfer of foreign genes into a variety of hosts including human cells. However, the genomic structure of any of the several known MuLV-A's has not been established and the evolutionary relationship of amphotropic retroviruses to the numerous exogenous or endogenous MuLV strains remains elusive. Herein we present a complete genetic structure of a novel amphotropic virus designated MuLV-1313 and demonstrate that this retrovirus together with other MuLV-A's belongs to a distinct molecular, biological and phylogenetic class among the MuLV strains isolated from a large number of the laboratory inbred or feral mice. RESULTS: The host range of MuLV-1313 is similar to the previously isolated MuLV-A's except that this virus replicates efficiently in mammalian as well as in chicken cells. Compared to ENV proteins of other MuLV-A's (4070A, 1504A and 10A-1), the gp70 protein of MuLV-1313 exhibits differences in its signal peptides and the proline-rich hinge regions. However, the MuLV-1313 envelope protein is totally unrelated to those present in a broad range of murine retroviruses that have been isolated from various inbred and feral mice globally. Genetic analysis of the entire MuLV-1313 genome by dot plot analyses, which compares each nucleotide of one genome with the corresponding nucleotide of another, revealed that the genome of this virus, with the exception of the env gene, is more closely related to the biologically distinct wild mouse ecotropic retrovirus (Cas-Br-E) isolated from another region of the Southern California, than to any of the 15 MuLV strains whose full-length sequences are present in the GenBank. This finding was corroborated by phylogenetic analyses and hierarchical clustering of the entire genomic sequence of MuLV-1313, which also placed all MULV-A's in a genetically distinct category among the large family of retroviruses isolated from numerous mouse strains globally. Likewise, construction of separate dendrograms for each of the Gag, Pol and Env proteins of MuLV-1313 demonstrated that the amphotropic retroviruses belong to a phylogenetically exclusive group of gammaretroviruses compared to all known MuLV strains. CONCLUSION: The molecular, biological and phylogenetic properties of amphotropic retroviruses including MuLV-1313 are distinct compared to a large family of exogenously- or endogenously-transmitted ecotropic, polytropic and xenotropic MuLV strains of the laboratory and feral mice. Further, both the naturally occurring amphotropic and a biologically discrete ecotropic retrovirus of the Southern California wild mice are more closely related to each other on the evolutionary tree than any other mammalian gammaretrovirus indicating a common origin of these viruses. This is the first report of a complete genomic analysis of a unique group of phylogenetically distinct amphotropic virus.
Assuntos
Evolução Molecular , Gammaretrovirus/genética , Genoma Viral/genética , Vírus da Leucemia Murina/classificação , Vírus da Leucemia Murina/genética , Filogenia , Animais , Linhagem Celular , Embrião de Galinha , DNA Viral/análise , Gammaretrovirus/classificação , Produtos do Gene env/química , Produtos do Gene env/genética , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene pol/química , Produtos do Gene pol/genética , Vírus da Leucemia Murina/patogenicidade , Camundongos , Dados de Sequência Molecular , Ratos , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
OBJECTIVES: The paper gives examples of the bioinformatics services provided in a variety of different libraries by librarians with a broad range of educational background and training. METHODS: Two investigators sent an email inquiry to attendees of the "National Center for Biotechnology Information's (NCBI) Introduction to Molecular Biology Information Resources" or "NCBI Advanced Workshop for Bioinformatics Information Specialists (NAWBIS)" courses. The thirty-five-item questionnaire addressed areas such as educational background, library setting, types and numbers of users served, and bioinformatics training and support services provided. Answers were compiled into program vignettes. DISCUSSION: The bioinformatics support services addressed in the paper are based in libraries with academic and clinical settings. Services have been established through different means: in collaboration with biology faculty as part of formal courses, through teaching workshops in the library, through one-on-one consultations, and by other methods. Librarians with backgrounds from art history to doctoral degrees in genetics have worked to establish these programs. CONCLUSION: Successful bioinformatics support programs can be established in libraries in a variety of different settings and by staff with a variety of different backgrounds and approaches.
Assuntos
Biologia Computacional/educação , Educação Continuada/métodos , Capacitação em Serviço/métodos , Bibliotecas , Serviços de Biblioteca , Serviços de Biblioteca/tendências , Desenvolvimento de Programas , Inquéritos e Questionários , Recursos HumanosRESUMO
Chemical principles dictate that the specific binding of a target to its complementary probes on a DNA microarray surface, and the nonspecific binding between other nucleotide segments and the same probes, are mutually competitive. We demonstrate that this mechanism can be understood by considering the competitive chemical reaction taking place on the microarray surface. Inspired by the pioneering work of Zhang and Hekstra, we have developed a physical model for microarray signal analysis, based on possible reaction mechanisms, and implemented it with a parallel, generic, simulated-annealing algorithm. Using data supplied by the Affymetrix Latin-square spike-in experiments, our model showed excellent fitting of the data. This correlation between the predicted expression levels and the spike-in concentrations of test transcripts demonstrated good predictive abilities of our model.
Assuntos
Algoritmos , Sondas de DNA/química , Modelos Químicos , Modelos Moleculares , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fatores de Transcrição/química , Simulação por Computador , Sondas de DNA/genética , Modelos Genéticos , Fatores de Transcrição/genéticaRESUMO
The glutathione S-transferase Mu class (GSTM) genes encode phase II metabolism enzymes that are involved in the detoxification of various carcinogens and drugs. Some genetic polymorphisms in GSTM genes are related to disease phenotypes and drug-metabolism differences in the population. Polymorphisms that alter gene-splicing patterns are functionally very important because they often lead to the insertion or deletion of many amino acids. To identify inter-individual differences in the splicing pattern of the GSTM4 gene, we used reverse transcriptase polymerase chain reaction (RT-PCR) to screen cDNA from 96 human liver samples. We discovered a novel splice variant of GSTM4 that resulted from tandem skipping of exons 4 and 5. This exon-skipping event is associated with a mutation at the splice acceptor site in intron 4.
Assuntos
Éxons , Glutationa Transferase/genética , Mutação Puntual , Splicing de RNA , DNA Complementar/genética , DNA Complementar/metabolismo , DNA Recombinante/metabolismo , Variação Genética , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Humanos , Íntrons , Fígado/enzimologia , Fígado/metabolismo , Modelos Genéticos , Polimorfismo Genético , Conformação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ab initio prediction of functional exon splicing enhancer (ESE) elements based on RNA sequences present a challenge in the evaluation of the functional impacts of human genetic polymorphisms on splicing. To better understand the behavior of ESEs, we studied their distribution in human exons and introns for four known SR protein-binding motifs: SF2/SAF, SC35, SRp40, and SRp55. ESEs are enriched in regions in exons that are close to the splice sites, especially in the region 80 to 120 bases away from the ends of splice acceptor sites. Significant enrichment of ESEs is associated with weak splice acceptor sites but not weak donor sites. ESE density decreases at the 3 ends of long exons. ESEs are also enriched in introns with weak donor or acceptor sites. These characteristics of ESEs may help to predict functional ESE sites in RNA sequences.
Assuntos
Processamento Alternativo , Éxons/genética , Sítios de Splice de RNA , Proteínas de Ligação a RNA/genética , Biologia Computacional , DNA Recombinante/química , Genoma Humano , Humanos , Íntrons/genética , Fatores de Transcrição/genéticaRESUMO
To examine the possibility that inter-individual differences in splicing partially explain the observed differences in CYP2D6 activity, we amplified its full-length cDNA in 96 human liver RNA samples and discovered five splice variants: intron 5 retention, intron 6 retention, intron 5 and intron 6 double retention, exon 3 skipping, and partial intron 1 retention. All of the CYP2D6 splice variants we identified are probably nonfunctional transcripts. Substantial inter-individual variation in the proportions of the CYP2D6 transcript represented by splice variants, measured by real-time PCR, suggests that the presence of these splice variants contributes to the population variation in CYP2D6 activity. Relatively high levels of intron 6 retention were not correlated with the newly discovered single nucleotide polymorphism 2988G > A in intron 6 (CYP2D6*41) but did correlate with the more common CYP2D6*34 allele. Our study prompts further investigations to explore the effect of these splice variants on drug metabolism.
Assuntos
Citocromo P-450 CYP2D6/genética , Fígado/enzimologia , Splicing de RNA , Sequência de Bases , Primers do DNA , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo ÚnicoRESUMO
OBJECTIVE: To explore new methods for the management of extensive deep partial thickness burn wounds. METHODS: Fifty burn patients with extensive deep partial thickness burn wounds were randomly divided into two groups: A and B groups. The patients in A group (n = 30) were treated with dermabrasion while those in B (n = 25) with conventional tangential excision. The first operation time in A and B groups was 12.3 +/- 10.7 hours and 47.2 +/- 11.5 hours, respectively. The patients' urine output, heart rate and arterial oxygen saturation (SaO2) were monitored. RESULTS: The mean one setting operation area in A and B groups were (65.5 +/- 19.4)% and (64.8 +/- 18.7)%, respectively. All the indices remained stable in both groups during and after the operation. Nevertheless, the burn wound healing time (20 days averagely) in A group was 10 days shorter than that in B group. The incidences of internal organ injury and bacteremia in A group were much lower than those in B group. Furthermore, the hospitalization cost in A group was decreased compared with that in B group. And the scar after wound healing was much less obvious in A group than that in B group. CONCLUSION: Dermabrasion during early postburn shock stage for the management of deep partial thickness burn wound had many advantages such as: easy manipulation, less injury to patients, lower infection rate, less complications and quicker burn wound healing.