RESUMO
Vernalization, a long-term cold-mediated acquisition of flowering competence, is critically regulated by VERNALIZATION INSENSITIVE 3 (VIN3), a gene induced by vernalization in Arabidopsis. Although the function of VIN3 has been extensively studied, how VIN3 expression itself is upregulated by long-term cold is not well understood. In this study, we identified a vernalization-responsive cis-element in the VIN3 promoter, VREVIN3, composed of a G-box and an evening element (EE). Mutations in either the G-box or the EE prevented VIN3 expression from being fully induced upon vernalization, leading to defects in the vernalization response. We determined that the core clock proteins CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE-ELONGATED HYPOCOTYL (LHY) associate with the EE of VREVIN3, both in vitro and in vivo. In a cca1 lhy double mutant background harboring a functional FRIGIDA allele, long-term cold-mediated VIN3 induction and acceleration of flowering were impaired, especially under mild cold conditions such as at 12°C. During prolonged cold exposure, oscillations of CCA1/LHY transcripts were altered, while CCA1 abundance increased at dusk, coinciding with the diurnal peak of VIN3 transcripts. We propose that modulation of the clock proteins CCA1 and LHY participates in the systems involved in sensing long-term cold for the activation of VIN3 transcription.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Ritmo Circadiano/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Hipocótilo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/ß-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Lactonas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Proteínas de Arabidopsis/genética , Domínio Catalítico/genética , Hidrólise , Mutação , Oryza/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas , Receptores de Superfície Celular/genéticaRESUMO
A growth technique to directly prepare two-dimensional (2D) materials onto conventional semiconductor substrates, enabling low-temperature, high-throughput, and large-area capability, is needed to realize competitive 2D transition-metal dichalcogenide (TMD)/three-dimensional (3D) semiconductor heterojunction devices. Therefore, we herein successfully developed an atmospheric-pressure plasma-enhanced chemical vapor deposition (AP-PECVD) technique, which could grow MoS2 and WS2 multilayers directly onto PET flexible substrate as well as 4-in. Si substrates at temperatures of <200 °C. The as-fabricated MoS2/Si and WS2/Si heterojunctions exhibited large and fast photocurrent responses under illumination of a green light. The measured photocurrent was linearly proportional to the laser power, indicating that trapping and detrapping of the photogenerated carriers at defect states could not significantly suppress the collection of photocarriers. All the results demonstrated that our AP-PECVD method could produce high-quality TMD/Si 2D-3D heterojunctions for optoelectronic applications.
RESUMO
A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Padronização Corporal/genética , Proteínas de Ligação a DNA/metabolismo , Flores/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Estabilidade de RNA/genética , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Mutação/genética , Fotoperíodo , Raízes de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
BACKGROUND: The level of loss of heterozygosity (LOH) that reduces a gene dose and exerts a cell-adverse effect is known to be a parameter for the genetic staging of gastric cancers. This study investigated if the cell-adverse effect induced with the gene reduction was a rate-limiting factor for the LOH events in two distinct histologic types of gastric cancers, the diffuse- and intestinal-types. METHODS: The pathologic specimens obtained from 145 gastric cancer patients were examined for the level of LOH using 40 microsatellite markers on eight cancer-associated chromosomes (3p, 4p, 5q, 8p, 9p, 13q, 17p and 18q). RESULTS: Most of the cancer-associated chromosomes were found to belong to the gene-poor chromosomes and to contain a few stomach-specific genes that were highly expressed. A baseline-level LOH involving one or no chromosome was frequent in diffuse-type gastric cancers. The chromosome 17 containing a relatively high density of genes was commonly lost in intestinal-type cancers but not in diffuse-type cancers. A high-level LOH involving four or more chromosomes tended to be frequent in the gastric cancers with intestinal and mixed differentiation. Disease relapse was common for gastric cancers with high-level LOH through both the hematogenous (38%) and non-hematogenous (36%) routes, and for the baseline-level LOH cases through the non-hematogenous route (67%). CONCLUSIONS: The cell-adverse effect of gene reduction is more tolerated in intestinal-type gastric cancers than in diffuse-type cancers, and the loss of high-dose genes is associated with hematogenous metastasis.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 17/genética , DNA de Neoplasias/genética , Perda de Heterozigosidade , Neoplasias Gástricas/genética , Biópsia , Feminino , Seguimentos , Heterozigoto , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Estudos Retrospectivos , Neoplasias Gástricas/patologiaRESUMO
Recent evidence suggests that gastric mucosal injury induces adaptive changes in DNA methylation. In this study, the methylation status of the key tissue-specific genes in normal gastric mucosa of healthy individuals and cancer patients was evaluated. The methylation-variable sites of 14 genes, including ulcer-healing genes (TFF1, TFF2, CDH1, and PPARG), were chosen from the CpG-island margins or non-island CpGs near the transcription start sites. The healthy individuals as well as the normal gastric mucosa of 23 ulcer, 21 non-invasive cancer, and 53 cancer patients were examined by semiquantitative methylation-specific polymerase chain reaction (PCR) analysis. The ulcer-healing genes were concurrently methylated with other genes depending on the presence or absence of CpG-islands in the normal mucosa of healthy individuals. Both the TFF2 and PPARG genes were frequently undermethylated in ulcer patients. The over- or intermediate-methylated TFF2 and undermethylated PPARG genes was more common in stage-1 cancer patients (71%) than in healthy individuals (10%; odds ratio [OR], 21.9) and non-invasive cancer patients (21%; OR, 8.9). The TFF2-PPARG methylation pattern of cancer patients was stronger in the older-age group (> or =55 yr; OR, 43.6). These results suggest that the combined methylation pattern of ulcer-healing genes serves as a sensitive marker for predicting cancer-prone gastric mucosa.
Assuntos
Metilação de DNA , Mucosa Gástrica , Neoplasias Gástricas , Úlcera Gástrica , Cicatrização/genética , Antígenos CD , Biomarcadores/metabolismo , Caderinas/genética , Ilhas de CpG , Feminino , Mucosa Gástrica/patologia , Mucosa Gástrica/fisiologia , Regulação Neoplásica da Expressão Gênica , Substâncias de Crescimento/genética , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , PPAR gama/genética , Peptídeos/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Úlcera Gástrica/genética , Úlcera Gástrica/patologia , Fator Trefoil-1 , Fator Trefoil-2 , Proteínas Supressoras de Tumor/genéticaRESUMO
CpG-island margins and non-island-CpG sites round the transcription start sites of CpG-island-positive and -negative genes are methylated to various degrees in a tissue-specific manner. These methylation-variable CpG sites were analyzed to delineate a relationship between the methylation and transcription of the tissue-specific genes. The level of tissue-specific transcription was estimated by counting the number of the total transcripts in the SAGE (serial analysis of gene expression) database. The methylation status of 12 CpG-island margins and 21 non-island CpG sites near the key tissue-specific genes was examined in pluripotent stromal cells obtained from fat and bone marrow samples as well as in lineage-committed cells from marrow bulk, stomach, colon, breast, and thyroid samples. Of the 33 CpG sites examined, 10 non-island-CpG sites, but none of the CpG-island margins were undermethylated concurrent with tissue-specific expression of their nearby genes. The net methylation of the 33 CpG sites and the net amount of non-island-CpG gene transcripts were high in stomach tissues and low in stromal cells. The present findings suggest that the methylation of the non-island-CpG sites is inversely associated with the expression of the nearby genes, and the concert effect of transitional-CpG methylation is linearly associated with the stomach-specific genes lacking CpG-islands.
Assuntos
Células-Tronco Adultas/metabolismo , Ilhas de CpG/genética , Metilação de DNA , Mucosa Gástrica/metabolismo , Tecido Adiposo/citologia , Adolescente , Adulto , Células-Tronco Adultas/citologia , Idoso , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Estômago/citologia , Células Estromais/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
The appropriate timing of flowering is pivotal for reproductive success in plants; thus, it is not surprising that flowering is regulated by complex genetic networks that are fine-tuned by endogenous signals and environmental cues. The Arabidopsis thaliana flowering-time gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) encodes a MADS box transcription factor and is one of the key floral activators integrating multiple floral inductive pathways, namely, long-day, vernalization, autonomous, and gibberellin-dependent pathways. To elucidate the downstream targets of SOC1, microarray analyses were performed. The analysis revealed that the soc1-2 knockout mutant has increased, and an SOC1 overexpression line has decreased, expression of cold response genes such as CBFs (for CRT/DRE binding factors) and COR (for cold regulated) genes, suggesting that SOC1 negatively regulates the expression of the cold response genes. By contrast, overexpression of cold-inducible CBFs caused late flowering through increased expression of FLOWERING LOCUS C (FLC), an upstream negative regulator of SOC1. Our results demonstrate the presence of a feedback loop between cold response and flowering-time regulation; this loop delays flowering through the increase of FLC when a cold spell is transient as in fall or early spring but suppresses the cold response when floral induction occurs through the repression of cold-inducible genes by SOC1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Temperatura Baixa , Flores/metabolismo , Flores/fisiologia , Proteínas de Domínio MADS/metabolismo , Proteínas de Domínio MADS/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Imunoprecipitação da Cromatina , Flores/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Domínio MADS/genética , Dados de Sequência Molecular , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix alphaC rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2alpha phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of alphaC and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of alphaC are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Substituição de Aminoácidos , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/genética , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: A loss of heterozygosity (LOH) represents a unilateral chromosomal loss that reduces the dose of highly repetitive Alu, L1, and LTR retroelements. The aim of this study was to determine if the LOH events can affect the spread of retroelement methylation in the 5'-end transitional area between the CpG islands and their nearest retroelements. METHODS: The 5'-transitional area of all human genes (22,297) was measured according to the nearest retroelements to the transcription start sites. For 50 gastric cancer specimens, the level of LOH events on eight cancer-associated chromosomes was estimated using the microsatellite markers, and the 5'-transitional CpGs of 20 selected genes were examined by methylation analysis using the bisulfite-modified DNA. RESULTS: The extent of the transitional area was significantly shorter with the nearest Alu elements than with the nearest L1 and LTR elements, as well as in the extragenic regions containing a higher density of retroelements than in the intragenic regions. The CpG islands neighbouring a high density of Alu elements were consistently hypomethylated in both normal and tumor tissues. The 5'-transitional methylated CpG sites bordered by a low density of Alu elements or the L1 and LTR elements were hypomethylated more frequently in the high-level LOH cases than in the low-level LOH cases. CONCLUSION: The 5'-transitional methylated CpG sites not completely protected by the Alu elements were hypomethylated in association with LOH events in gastric cancers. This suggests that an irreversible unbalanced decrease in the genomic dose reduces the spread of L1 methylation in the 5'-end regions of genes.
Assuntos
Região 5'-Flanqueadora/genética , Ilhas de CpG , Metilação de DNA , DNA de Neoplasias/genética , Perda de Heterozigosidade , Retroelementos , Neoplasias Gástricas/genética , Idoso , Elementos Alu , DNA de Neoplasias/isolamento & purificação , Mecanismo Genético de Compensação de Dose , Epigênese Genética , Feminino , Mucosa Gástrica/química , Dosagem de Genes , Heterocromatina/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Neoplasias Gástricas/química , Neoplasias Gástricas/patologia , Sequências Repetidas TerminaisRESUMO
The stage of gastrointestinal cancers has been correlated with the loss of heterozygosity (LOH) and the presence of microsatellite instability (MSI). This study delineated the category of the extent of LOH and the presence of MSI for the genetic classification of the intestinal-type and diffuse-type gastric cancers that frequently exhibited intralesional heterogeneity. A total of 390 tumor foci from 116 gastric cancers were screened using a panel of 40 microsatellite markers on chromosomes 3p, 4p, 5q, 8p, 9p, 13q, 17p, and 18q. One MSI-positive gastric cancer accompanying a LOH-positive focus and 19 gastric cancers with an intralesional LOH heterogeneity with a similar extent were identified. One hundred and sixteen gastric cancers were categorized based on the presence of MSI (16 cases) and the extent of LOH (100 cases) in a representative focus. A large fraction of MSI-positive cases was found in the intestinal-type (94%), late-onset (mean age 68 years), early-stage (75%) diseases (P<0.05). The diffuse-type gastric cancers with a baseline-level loss involving zero or one chromosome showed a correlation with the earlier onset (mean age 45 years), advanced-stage (81%) diseases (P<0.0001). In both the intestinal-type and diffuse-type gastric cancers, a low-level loss involving 0-3 chromosomes (2-3 chromosomes in the diffuse type) and a high-level loss involving 4-7 chromosomes were predominant in the early (69%) and advanced (86%) stages, respectively (P<0.0001), at similar mean ages of onset (61 years and 65 years). Gastric cancers were categorized into low-risk (MSI and low-level LOH) and high-risk (baseline-level and high-level LOH) genotypes displaying cell-type- and age-dependent oncogenicity.
Assuntos
Instabilidade Genômica , Perda de Heterozigosidade , Repetições de Microssatélites/genética , Neoplasias Gástricas/genética , Adulto , Idoso , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/classificação , Neoplasias Gástricas/patologiaRESUMO
We investigated beta-catenin and adenomatous polyposis coli (APC) gene abnormalities in human pilomatricoma, in which a high incidence of beta-catenin gene mutations has been reported. Nucleated tumour cells were microdissected from 20 paraffin-embedded pilomatricomas. Exon 3 of the beta-catenin gene was amplified using polymerase chain reaction and sequencing analysis was performed. Immunostaining for beta-catenin and lymphoid-enhancer factor-1 was performed using the avidin-biotin-peroxidase method. Dinucleotide repeat markers D5S409 and D5S299 were used for polymerase chain reaction-based microsatellite analysis of the APC gene. The mutation cluster region of the APC gene was amplified using polymerase chain reaction and sequenced. Sequencing analysis revealed beta-catenin gene mutations in 30%. All studied samples showed nuclear lymphoid-enhancer factor-1 and cytoplasmic/nuclear beta-catenin expression. Loss of heterozygosity was observed in the APC gene, but no mutations in the mutation cluster region were found in seven tumours without beta-catenin mutations. The frequency of beta-catenin gene mutations was remarkably low, thus suggesting (i) the presence of mutations in other than exon 3 of the beta-catenin gene, (ii) a possible role of APC gene abnormalities, or (iii) involvement of other components of the Wingless-type MMTV integration site family pathway.