RESUMO
Development in multicellular organisms requires the coordinated production of a large number of specialized cell types through sophisticated signaling mechanisms. Non-cell-autonomous signals are one of the key mechanisms by which organisms coordinate development. In plants, intercellular movement of transcription factors and other mobile signals, such as hormones and peptides, is essential for normal development. Through a combination of different approaches, a large number of non-cell-autonomous signals that control plant development have been identified. We review some of the transcriptional regulators that traffic between cells, as well as how changes in symplasmic continuity affect and are affected by development. We also review current models for how mobile signals move via plasmodesmata and how movement is inhibited. Finally, we consider challenges in and new tools for studying protein movement.
Assuntos
Comunicação Celular/fisiologia , Desenvolvimento Vegetal/fisiologia , Proteínas de Plantas/metabolismo , Plasmodesmos/fisiologia , Transporte Proteico/fisiologia , Parede Celular/ultraestrutura , Cloroplastos/fisiologia , Florígeno , Glucanos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Plasmodesmos/ultraestrutura , RNA de Plantas/fisiologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Tricomas/metabolismoRESUMO
Serratia sp. ATCC 39006 produces intracellular gas vesicles to enable upward flotation in water columns. It also uses flagellar rotation to swim through liquid and swarm across semi-solid surfaces. Flotation and motility can be co-regulated with production of a ß-lactam antibiotic (carbapenem carboxylate) and a linear tripyrrole red antibiotic, prodigiosin. Production of gas vesicles, carbapenem and prodigiosin antibiotics, and motility are controlled by master transcriptional and post-transcriptional regulators, including the SmaI/SmaR-based quorum sensing system and the mRNA binding protein, RsmA. Recently, the ribose operon repressor, RbsR, was also defined as a pleiotropic regulator of flotation and virulence factor elaboration in this strain. Here, we report the discovery of a new global regulator (FloR; a DeoR family transcription factor) that modulates flotation through control of gas vesicle morphogenesis. The floR mutation is highly pleiotropic, down-regulating production of gas vesicles, carbapenem and prodigiosin antibiotics, and infection in Caenorhabditis elegans, but up-regulating flagellar motility. Detailed proteomic analysis using TMT peptide labelling and LC-MS/MS revealed that FloR is a physiological master regulator that operates through subordinate pleiotropic regulators including Rap, RpoS, RsmA, PigU, PstS and PigT.
Assuntos
Proteínas de Bactérias/metabolismo , Serratia , Virulência/genética , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Carbapenêmicos/biossíntese , Cromatografia Líquida , Regulação Bacteriana da Expressão Gênica , Mutação , Óperon , Prodigiosina/biossíntese , Proteômica , Percepção de Quorum , Serratia/genética , Serratia/metabolismo , Serratia/patogenicidade , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismoRESUMO
Both endogenous plant proteins and viral movement proteins associate with microtubules to promote their movement through plasmodesmata. The association of viral movement proteins with microtubules facilitates the formation of virus-associated replication complexes, which are required for the amplification and subsequent spread of the virus. However, the role of microtubules in the intercellular movement of plant proteins is less clear. Here we show that the SHORT-ROOT (SHR) protein, which moves between cells in the root to regulate root radial patterning, interacts with a type-14 kinesin, KINESIN G (KinG). KinG is a calponin homology domain kinesin that directly interacts with the SHR-binding protein SIEL (SHR-INTERACING EMBRYONIC LETHAL) and localizes to both microtubules and actin. Since SIEL and SHR associate with endosomes, we suggest that KinG serves as a linker between SIEL, SHR, and the plant cytoskeleton. Loss of KinG function results in a decrease in the intercellular movement of SHR and an increase in the sensitivity of SHR movement to treatment with oryzalin. Examination of SHR and KinG localization and dynamics in live cells suggests that KinG is a nonmotile kinesin that promotes the pausing of SHR-associated endosomes. We suggest a model in which interaction of KinG with SHR allows for the formation of stable movement complexes that facilitate the cell-to-cell transport of SHR.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Espaço Extracelular/metabolismo , Espaço Intracelular/metabolismo , Cinesinas/metabolismo , Fatores de Transcrição/metabolismo , Actinas/metabolismo , Proteínas de Arabidopsis/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dinitrobenzenos/farmacologia , Endossomos/metabolismo , Cinesinas/química , Meristema/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Mutação/genética , Epiderme Vegetal/citologia , Folhas de Planta/citologia , Raízes de Plantas/metabolismo , Domínios Proteicos , Transporte Proteico , Especificidade da Espécie , Frações Subcelulares/metabolismo , Sulfanilamidas/farmacologia , Tiazolidinas/farmacologia , Nicotiana/citologiaRESUMO
Eukaryotic circadian clocks utilize the ubiquitin proteasome system to precisely degrade clock proteins. In plants, the F-box-type E3 ubiquitin ligases ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1), and LOV KELCH PROTEIN2 (LKP2) regulate clock period and couple the clock to photoperiodic flowering in response to end-of-day light conditions. To better understand their functions, we expressed decoy ZTL, FKF1, and LKP2 proteins that associate with target proteins but are unable to ubiquitylate their targets in Arabidopsis (Arabidopsis thaliana). These dominant-negative forms of the proteins inhibit the ubiquitylation of target proteins and allow for the study of ubiquitylation-independent and -dependent functions of ZTL, FKF1, and LKP2. We demonstrate the effects of expressing ZTL, FKF1, and LKP2 decoys on the circadian clock and flowering time. Furthermore, the decoy E3 ligases trap substrate interactions, and using immunoprecipitation-mass spectrometry, we identify interacting partners. We focus studies on the clock transcription factor CCA1 HIKING EXPEDITION (CHE) and show that ZTL interacts directly with CHE and can mediate CHE ubiquitylation. We also demonstrate that CHE protein is degraded in the dark and that degradation is reduced in a ztl mutant plant, showing that CHE is a bona fide ZTL target protein. This work increases our understanding of the genetic and biochemical roles for ZTL, FKF1, and LKP2 and also demonstrates an effective methodology for studying complicated genetic redundancy among E3 ubiquitin ligases.
Assuntos
Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/fisiologia , Proteínas Repressoras/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Domínios Proteicos , Mapas de Interação de Proteínas , Proteínas Repressoras/genética , UbiquitinaçãoRESUMO
Formation of specialized cells and tissues at defined times and in specific positions is essential for the development of multicellular organisms. Often this developmental precision is achieved through intercellular signaling networks, which establish patterns of differential gene expression and ultimately the specification of distinct cell fates. Here we address the question of how the Short-root (SHR) proteins from Arabidopsis thaliana (AtSHR), Brachypodium distachyon (BdSHR), and Oryza sativa (OsSHR1 and OsSHR2) function in patterning the root ground tissue. We find that all of the SHR proteins function as mobile signals in A. thaliana and all of the SHR homologs physically interact with the AtSHR binding protein, Scarecow (SCR). Unlike AtSHR, movement of the SHR homologs was not limited to the endodermis. Instead, the SHR proteins moved multiple cell layers and determined the number of cortex, not endodermal, cell layers formed in the root. Our results in A. thaliana are consistent with a mechanism by which the regulated movement of the SHR transcription factor determines the number of cortex cell layers produced in the roots of B. distachyon and O. sativa. These data also provide a new model for ground tissue patterning in A. thaliana in which the ability to form a functional endodermis is spatially limited independently of SHR.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brachypodium/metabolismo , Oryza/metabolismo , Raízes de Plantas/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Brachypodium/genética , Oryza/genética , Fatores de Transcrição/genéticaRESUMO
Exposure of Arabidopsis thaliana plants to low non-freezing temperatures results in an increase in freezing tolerance that involves action of the C-repeat binding factor (CBF) regulatory pathway. CBF1, CBF2 and CBF3, which are rapidly induced in response to low temperature, encode closely related AP2/ERF DNA-binding proteins that recognize the C-repeat (CRT)/dehydration-responsive element (DRE) DNA regulatory element present in the promoters of CBF-regulated genes. The CBF transcription factors alter the expression of more than 100 genes, known as the CBF regulon, which contribute to an increase in freezing tolerance. In this study, we investigated the extent to which cold induction of the CBF regulon is regulated by transcription factors other than CBF1, CBF2 and CBF3, and whether freezing tolerance is dependent on a functional CBF-CRT/DRE regulatory module. To address these issues we generated transgenic lines that constitutively overexpressed a truncated version of CBF2 that had dominant negative effects on the function of the CBF-CRT/DRE regulatory module, and 11 transcription factors encoded by genes that were rapidly cold-induced in parallel with the 'first-wave' CBF genes, and determined the effects that overexpressing these proteins had on global gene expression and freezing tolerance. Our results indicate that cold regulation of the CBF regulon involves extensive co-regulation by other first-wave transcription factors; that the low-temperature regulatory network beyond the CBF pathway is complex and highly interconnected; and that the increase in freezing tolerance that occurs with cold acclimation is only partially dependent on the CBF-CRT/DRE regulatory module.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Regulon/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismoRESUMO
The natural range of Arabidopsis thaliana (Arabidopsis) encompasses geographical regions that have greatly differing local climates, including harshness of winter temperatures. A question thus raised is whether differences in freezing tolerance might contribute to local adaptation in Arabidopsis. Consistent with this possibility is that Arabidopsis accessions differ in freezing tolerance and that those collected from colder northern latitudes are generally more tolerant to freezing than those collected from warmer southern latitudes. Moreover, recent studies with Arabidopsis genotypes collected from sites in Sweden (SW) and Italy (IT) have established that the two accessions are locally adapted, that the SW ecotype is more tolerant of freezing than the IT ecotype, and that genetic differences between the two ecotypes that condition local adaptation and freezing tolerance map to a region that includes the C-repeat binding factor (CBF) locus. The CBF locus includes three genes - CBF1, CBF2 and CBF3 - that are induced by low temperature and encode transcription factors that regulate a group of more than 100 genes, the CBF regulon, which impart freezing tolerance. Here we show that cold induction of most CBF regulon genes is lower in IT plants compared with SW plants, and that this is due to the IT CBF2 gene encoding a non-functional CBF2 protein. The non-functional IT CBF2 protein also contributes to the lower freezing tolerance of the IT plants compared with the SW plants. Taken together, studies on the SW and IT ecotypes provide evidence that natural variation in the CBF pathway has contributed to adaptive evolution in these Arabidopsis populations.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Temperatura Baixa , Variação Genética , Transativadores/genética , Fatores de Transcrição/genética , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Arabidopsis/classificação , Ecótipo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Geografia , Itália , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Locos de Características Quantitativas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Especificidade da Espécie , SuéciaRESUMO
The CBF (C-repeat binding factor) pathway has a major role in plant cold acclimation, the process whereby certain plants increase in freezing tolerance in response to low nonfreezing temperatures. In Arabidopsis thaliana, the pathway is characterized by rapid cold induction of CBF1, CBF2, and CBF3, which encode transcriptional activators, followed by induction of CBF-targeted genes that impart freezing tolerance. At warm temperatures, CBF transcript levels are low, but oscillate due to circadian regulation with peak expression occurring at 8 h after dawn (zeitgeber time 8; ZT8). Here, we establish that the CBF pathway is also regulated by photoperiod at warm temperatures. At ZT8, CBF transcript levels in short-day (SD; 8-h photoperiod) plants were three- to fivefold higher than in long-day plants (LD; 16-h photoperiod). Moreover, the freezing tolerance of SD plants was greater than that of LD plants. Genetic analysis indicated that phytochrome B (PHYB) and two phytochrome-interacting factors, PIF4 and PIF7, act to down-regulate the CBF pathway and freezing tolerance under LD conditions. Down-regulation of the CBF pathway in LD plants correlated with higher PIF4 and PIF7 transcript levels and greater stability of the PIF4 and PIF7 proteins under LD conditions. Our results indicate that during the warm LD growing season, the CBF pathway is actively repressed by PHYB, PIF4, and PIF7, thus mitigating allocation of energy and nutrient resources toward unneeded frost protection. This repression is relieved by shortening day length resulting in up-regulation of the CBF pathway and increased freezing tolerance in preparation for coming cold temperatures.
Assuntos
Aclimatação/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Congelamento , Regulação da Expressão Gênica de Plantas/fisiologia , Fotoperíodo , Transativadores/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Imunoprecipitação da Cromatina , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Immunoblotting , Fitocromo B/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1-JAZ-DELLA-PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated.
Assuntos
Ciclopentanos/metabolismo , Giberelinas/metabolismo , Oxilipinas/metabolismo , Plantas/metabolismo , Transdução de Sinais/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclopentanos/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Giberelinas/farmacologia , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Oxilipinas/farmacologia , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Ligação Proteica , Proteólise/efeitos dos fármacos , Interferência de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Four repetitive element sequence-based polymerase chain reaction (rep-PCR) methods, namely repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), polytrinucleotide (GTG)5 -PCR and BOX-PCR, were evaluated for the molecular differentiation of 12 probiotic Lactobacillus strains previously isolated from the gastrointestinal tract of chickens and used as a multistrain probiotic. This study represents the first analysis of the comparative efficacy of these four rep-PCR methods and their combination (composite rep-PCR) in the molecular typing of Lactobacillus strains based on a discriminatory index (D). RESULTS: Species-specific and strain-specific profiles were observed from rep-PCR. From the numerical analysis of composite rep-PCR, BOX-PCR, (GTG)5 -PCR, REP-PCR and ERIC-PCR, D values of 0.9118, 0.9044, 0.8897, 0.8750 and 0.8529 respectively were obtained. Composite rep-PCR analysis was the most discriminative method, with eight Lactobacillus strains, namely L. brevis ATCC 14869(T) , L. reuteri C 10, L. reuteri ATCC 23272(T) , L. gallinarum ATCC 33199(T) , L. salivarius ATCC 11741(T) , L. salivarius I 24, L. panis JCM 11053(T) and L. panis C 17, being differentiated at the strain level. CONCLUSION: Composite rep-PCR analysis is potentially a useful fingerprinting method to discriminate probiotic Lactobacillus strains isolated from the gastrointestinal tract of chickens.
Assuntos
DNA Bacteriano/metabolismo , Dieta/veterinária , Lactobacillus/classificação , Probióticos/classificação , Animais , Galinhas/microbiologia , Impressões Digitais de DNA/veterinária , Trato Gastrointestinal/microbiologia , Mucosa Intestinal/microbiologia , Sequências Repetidas Invertidas , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Malásia , Tipagem Molecular/métodos , Tipagem Molecular/veterinária , Filogenia , Reação em Cadeia da Polimerase/veterinária , Probióticos/isolamento & purificação , Probióticos/metabolismo , Sequências Repetitivas de Ácido Nucleico , Especificidade da EspécieRESUMO
Cdc25A is a dual specificity protein phosphatase that activates cyclin/cyclin-dependent protein kinase (Cdk) complexes by removing inhibitory phosphates from conserved threonine and tyrosine in Cdks. To address how Cdc25A promotes apoptosis, Jurkat cells were treated with staurosporine, an apoptosis inducer. Upon staurosporine treatment, a Cdc25A C-terminal 37-kDa fragment, designated C37, was generated by caspase cleavage at Asp-223. Thr-507 in C37 became dephosphorylated, which prevented 14-3-3 binding, as shown previously. C37 exhibited higher phosphatase activity than full-length Cdc25A. C37 with alanine substitution for Thr-507 (C37/T507A) that imitated the cleavage product during staurosporine treatment interacted with Cdc2, Cdk2, cyclin A, and cyclin B1 and markedly activated cyclin B1/Cdc2. The dephosphorylation of Thr-507 might expose the Cdc2/Cdk2-docking site in C37. C37/T507A also induced apoptosis in Jurkat and K562 cells, resulting from activating cyclin B1/Cdc2 but not Cdk2. Thus, this study reveals that Cdc25A is a pro-apoptotic protein that amplifies staurosporine-induced apoptosis through the activation of cyclin B1/Cdc2 by its C-terminal domain.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B1/metabolismo , Fosfatases cdc25/metabolismo , Apoptose , Caspases/metabolismo , Glutationa Transferase/metabolismo , Humanos , Células Jurkat , Células K562 , Modelos Biológicos , Mutagênese Sítio-Dirigida , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Ubiquitina/químicaRESUMO
ZEITLUPE (ZTL), a photoreceptor with E3 ubiquitin ligase activity, communicates end-of-day light conditions to the plant circadian clock. It still remains unclear how ZTL protein accumulates in the light but does not destabilize target proteins before dusk. Two deubiquitylating enzymes, UBIQUITIN-SPECIFIC PROTEASE 12 and 13 (UBP12 and UBP13), which regulate clock period and protein ubiquitylation in a manner opposite to ZTL, associate with the ZTL protein complex. Here we demonstrate that the ZTL interacting partner, GIGANTEA (GI), recruits UBP12 and UBP13 to the ZTL photoreceptor complex. We show that loss of UBP12 and UBP13 reduces ZTL and GI protein levels through a post-transcriptional mechanism. Furthermore, a ZTL target protein is unable to accumulate to normal levels in ubp mutants. This demonstrates that the ZTL photoreceptor complex contains both ubiquitin-conjugating and -deconjugating enzymes, and that these two opposing enzyme types are necessary for circadian clock pacing. This shows that deubiquitylating enzymes are a core element of circadian clocks, conserved from plants to animals.
Assuntos
Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/fisiologia , Endopeptidases/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Endopeptidases/genética , Regulação da Expressão Gênica de Plantas , Mutação , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
The circadian clock relies on regulated degradation of clock proteins to maintain rhythmicity. Despite this, we know few components that mediate protein degradation. This is due to high levels of functional redundancy within plant E3 ubiquitin ligase families. In order to overcome this issue and discover E3 ubiquitin ligases that control circadian function, we generated a library of transgenic Arabidopsis plants expressing dominant-negative 'decoy' E3 ubiquitin ligases. We determined their effects on the circadian clock and identified dozens of new potential regulators of circadian function. To demonstrate the potency of the decoy screening methodology to overcome redundancy and identify bona fide clock regulators, we performed follow-up studies on MAC3A (PUB59) and MAC3B (PUB60). We show that they redundantly control circadian period by regulating splicing. This work demonstrates the viability of ubiquitin ligase decoys as a screening platform to overcome genetic challenges and discover E3 ubiquitin ligases that regulate plant development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Relógios Circadianos , Regulação da Expressão Gênica de Plantas , Testes Genéticos/métodos , Ubiquitina-Proteína Ligases/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Plantas Geneticamente Modificadas , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
The copy numbers of 16S rRNA genes in 12 probiotic Lactobacillus strains of poultry origin were analyzed. Genomic DNA of the strains was digested with restriction endonucleases that do not cut within the 16S rRNA gene of the strains. This was followed by Southern hybridization with a biotinylated probe complementary to the 16S rRNA gene. The copy number of the 16S rRNA gene within a Lactobacillus species was found to be conserved. From the hybridization results, Lactobacillus salivarius I 24 was estimated to have seven copies of the 16S rRNA gene, Lactobacillus panis C 17 to have five copies and Lactobacillus gallinarum strains I 16 and I 26 four copies. The 16S rRNA gene copy numbers of L. gallinarum and L. panis reported in the present study are the first record. Lactobacillus brevis strains I 12, I 23, I 25, I 211, I 218 and Lactobacillus reuteri strains C 1, C 10, C 16 were estimated to have at least four copies of the 16S rRNA gene. In addition, distinct rRNA restriction patterns which could discriminate the strains of L. reuteri and L. gallinarum were also detected. Information on 16S rRNA gene copy number is important for physiological, evolutionary and population studies of the bacteria.
Assuntos
Galinhas/microbiologia , Trato Gastrointestinal/microbiologia , Dosagem de Genes , Lactobacillus/genética , Probióticos , RNA Ribossômico 16S/genética , Animais , Southern Blotting , Enzimas de Restrição do DNA/metabolismo , Lactobacillus/classificação , Lactobacillus/isolamento & purificaçãoRESUMO
Failure of hollow trees in urban areas is a worldwide concern, and it can be caused by different mechanisms, i.e. bending stresses or flattening-related failures. Here we derive a new analytical expression for predicting the bending moment for tangential cracking, and compare the breaking moment of various failure modes, including Brazier buckling, tangential cracking, shear failure and conventional bending failure, as a function of t/R ratio, where t and R are the trunk wall thickness and trunk radius, respectively, of a hollow tree. We use Taiwan red cypress as an example and show that its failure modes and the corresponding t/R ratios are: Brazier buckling (Mode I), tangential cracking followed by longitudinal splitting (Mode II) and conventional bending failure (Mode III) for 0 < t/R < 0.06, 0.06 < t/R < 0.27 and 0.27 < t/R < 1, respectively. The exact values of those ratios may vary within and among species, but the variation is much smaller than individual mechanical properties. Also, shear failure, another type of cracking due to maximum shear stress near the neutral axis of the tree trunk, is unlikely to occur since it requires much larger bending moments. Hence, we conclude that tangential cracking due to cross-sectional flattening, followed by longitudinal splitting, is dominant for hollow trunks. Our equations are applicable to analyse straight hollow tree trunks and plant stems, but are not applicable to those with side openings or those with only heart decay. Our findings provide insights for those managing trees in urban situations and those managing for conservation of hollow-dependent fauna in both urban and rural settings.
RESUMO
The mapping of protein-protein interaction (PPI) networks and their dynamics are crucial steps to deciphering the function of a protein and its role in cellular pathways, making it critical to have comprehensive knowledge of a protein's interactome. Advances in affinity purification and mass spectrometry technology (AP-MS) have provided a powerful and unbiased method to capture higher-order protein complexes and decipher dynamic PPIs. However, the unbiased calling of nonspecific interactions and the ability to detect transient interactions remains challenging when using AP-MS, thereby hampering the detection of biologically meaningful complexes. Additionally, there are plant-specific challenges with AP-MS, such as a lack of protein-specific antibodies, which must be overcome to successfully identify PPIs. Here we discuss and describe a protocol designed to bypass the traditional challenges of AP-MS and provide a roadmap to identify bona fide PPIs in plants.
Assuntos
Cromatografia de Afinidade/métodos , Mapeamento de Interação de Proteínas/métodos , Espectrometria de Massas/métodos , Proteômica/métodosRESUMO
Distributions of growth strains in branches, straight trunks and basal sweeping trunks of Chamaecyparis formosensis Matsum. trees were measured with strain gauges. Microfibril angles (MFAs) of the S2 layer of the cell wall were measured by the iodine deposition method and their relationships with growth strain examined. The magnitude of the compressive stress on the lower side of trunks with a basal sweep was greater than that of the tensile stress at the surface of straight trunks. However, transverse compressive stress was similar around the trunk regardless of whether normal wood or compression wood was present. The released surface growth strains varied with MFA. At MFAs of 20-25 degrees , growth stress changed from tension to compression, and compressive stress increased dramatically in the compression wood region. Branches suffer bending stress due to self-loading. This stress is superimposed on the growth stress. Growth strains on the upper or lower sides of branches were larger than those in the trunks, suggesting that generation of growth stress on the lower sides of branches with extensive compression wood is affected by the gravitational bending stress due to self-loading. We conclude that branch form is affected by the interaction between the bending moment due to self-loading and that due to the asymmetric distribution of growth stress. Growth strain distribution in a branch differed depending on whether the branch was horizontal, upward bending or downward bending.
Assuntos
Chamaecyparis/anatomia & histologia , Chamaecyparis/crescimento & desenvolvimento , Árvores/anatomia & histologia , Árvores/crescimento & desenvolvimento , Fenômenos BiomecânicosRESUMO
Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.
Assuntos
Archaea/efeitos dos fármacos , Biblioteca Gênica , Variação Genética , Metano/metabolismo , Proantocianidinas/farmacologia , Rúmen/microbiologia , Animais , Archaea/classificação , Archaea/genética , Archaea/isolamento & purificação , Bovinos , DNA Arqueal/análise , DNA Ribossômico/análise , Genes de RNAr , Methanobacteriales/classificação , Methanobacteriales/genética , Methanobacteriales/isolamento & purificação , Methanomicrobiales/classificação , Methanomicrobiales/genética , Methanomicrobiales/isolamento & purificação , Methanosarcinales/classificação , Methanosarcinales/genética , Methanosarcinales/isolamento & purificação , Dados de Sequência Molecular , Proantocianidinas/metabolismo , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Among the major proteins in the rat seminal vesicle secretion, transglutaminase catalyzed the cross-links among RSVS I-III. Six peptide sequences determined from the trypsin digests of RSVS III were confirmed in the protein sequence derived from two paralogs, RSVS III(alpha) and RSVS III(beta) gene, in rat 3q42. Their transcription units are organized with the first exon encoding a signal peptide, and the second a secreted protein, whereas the third encompasses a 3'-non-translated nucleotide that shares common features of rapidly evolving substrates of transglutaminase (REST) gene family. These two genes have 99% identity in their coding region and both express in adult rats with the same transglutaminase cross-linking site, manifesting functional preservation. All of REST genes reported thus far for human and Muridae were mapped in a chromosomal locus between KCNS1 and SLPI suggesting the locus as an active evolving region. The molecular evolution of this gene family is discussed.