Soybean 14-3-3 gene family: identification and molecular characterization

The 14-3-3s are a group of proteins that are ubiquitously found in eukaryotes. Plant 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Recent studies in Arabidopsis and rice have demonstrated the isoform specificity in 14-3-3s and their client protein interactions. However, detailed characterization of 14-3-3 gene family in legumes has not been reported. In this study, soybean 14-3-3 proteins were identified and their molecular characterization performed. Data mining of soybean genome and expressed sequence tag databases identified 18 14-3-3 genes, of them 16 are transcribed. All 16 SGF14s have higher expression in embryo tissues suggesting their potential role in seed development. Subcellular localization of all transcribed SGF14s demonstrated that 14-3-3 proteins in soybean have isoform specificity, however, some overlaps were also observed between closely related isoforms. A comparative analysis of SGF14s with Arabidopsis and rice 14-3-3s indicated that SGF14s also group into epsilon and non-epsilon classes. However, unlike Arabidopsis and rice 14-3-3s, SGF14s contained only one kind of gene structure belonging to each class. Overall, soybean consists of the largest family of 14-3-3 proteins characterized to date. Our results provide a solid framework for further investigations into the role of SGF14s and their involvement in legume-specific functions.     

Gene expression patterns of the ALP family during zebrafish development

The actinin-associated LIM protein (ALP) genes belong to the PDZ/LIM protein family which is characterized by the presence of both a PDZ and a LIM domain. The ALP subfamily in mammals has four members: ALP, Elfin, Mystique and RIL. In this study, we have annotated and cloned the zebrafish ALP gene family and identified a zebrafish-specific fifth member of the family, the alp-like gene. We compared the zebrafish sequences to their human and mouse orthologues. A phylogenetic analysis based on the amino acid sequences showed the overall high degree of conservation within the family. We describe here the expression patterns for all five ALP family genes during zebrafish development. Whole mount in situ hybridization results revealed common and distinct expression patterns for the five genes. With the exception of elfin, all genes were expressed as maternal RNAs at early developmental stages. Gene expression for all of them appeared regulated and localized in specific regions at the eight different developmental stages studied. Expression for all five genes was observed in the central nervous system (CNS), which led us to further investigate brain-specific expression in sections of embryos at 2 days of development. In summary, we identified the zebrafish orthologues of the ALP family and determined their gene expression patterns during zebrafish embryogenesis. Finally, we compare our results to the limited expression data available for this gene family during mammalian development.     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3zeta and Bm14-3-3epsilon). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3zeta shared 90% identity with that in Drosophila, while Bm14-3-3epsilon shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3zeta expression levels are higher than Bm14-3-3epsilon in seven tissues and in four silkworm developmental stages examined. Bm14-3-3zeta was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3epsilon expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3zeta expression is universal and continuous, while Bm14-3-3epsilon expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.     

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori)

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3ζ and Bm14-3-3ε). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3ζ shared 90% identity with that in Drosophila, while Bm14-3-3ε shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3ζ expression levels are higher than Bm14-3-3ε in seven tissues and in four silkworm developmental stages examined. Bm14-3-3ζ was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3ε expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3ζ expression is universal and continuous, while Bm14-3-3ε expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.     

Expression of the dnmt3 genes in zebrafish development: similarity to Dnmt3a and Dnmt3b

The zebrafish differs from mammals in that they have six dnmt3 genes as opposed to the two that can produce a catalytically active protein in mammals. Zebrafish also do not show evidence of genomic imprinting and lack the Dnmt3l gene necessary to that process in mammals. As such, they offer a unique opportunity to compare the two genetic situations in order to define the roles of the multiple genes in developmental gene methylation. To this end, we have analyzed the developmental expression of the six dnmt3 genes in zebrafish and find that they fall into two distinct patterns. The expression patterns of the dnmt6 and dnmt8 genes, which more closely resemble the mammalian Dnmt3a gene in sequence, also show an expression pattern that is more similar to the expression of Dnmt3a rather than Dnmt3b. Conversely, the other four dnmt3 genes in zebrafish (dnmt3, dnmt4, dnmt5, and dnmt7) show an expression pattern that is more similar to Dnmt3b. The dnmt6 and dnmt8 genes are also expressed in the adult zebrafish and in the brain in particular. In situ expression analyses show that the dnmt6 and/or dnmt8 genes also show tissue-specific differences in expression with those two genes being more ubiquitously expressed in the developing zebrafish than the other dnmt3 genes. Although differences in dnmt3 function may exist between mammals and fish, our results showing similar expression patterns between the genes in fish and mammals suggest that the six dnmt3 genes in the zebrafish may be analogous to the two Dnmt3 genes in mammals.     

Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.     

Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs.     

Evolution and isoform specificity of plant 14-3-3 proteins

The 14-3-3 proteins, once thought of as obscure mammalian brain proteins, are fast becoming recognized as major regulators of plant primary metabolism and of other cellular processes. Their presence as large gene families in plants underscores their essential role in plant physiology. We have examined the Arabidopsis thaliana 14-3-3 gene family, which currently is the largest and most complete 14-3-3 family with at least 12 expressed members and 15 genes from the now completed Arabidopsis thaliana genome project. The phylogenetic branching of this family serves as the prototypical model for comparison with other large plant 14-3-3 families and as such may serve to rationalize clustering in a biological context. Equally important for ascribing common functions for the various 14-3-3 isoforms is determining an isoform-specific correlation with localization and target partnering. A summary of localization information available in the literature is presented. In an effort to identify specific 14-3-3 isoform location and participation in cellular processes, we have produced a panel of isoform-specific antibodies to Arabidopsis thaliana 14-3-3s and present initial immunolocalization studies that suggest biologically relevant, discriminative partnering of 14-3-3 isoforms.     

Cloning, expression, and chromosomal mapping of the human 14-3-3gamma gene (YWHAG) to 7q11.23.

The 14-3-3 family of proteins exerts diverse influences on the signal transduction pathways of cells. We have newly identified a human cDNA encoding the gamma subtype of the 14-3-3 family of genes. The deduced amino acid sequence of human 14-3-3gamma was identical to that of rat 14-3-3gamma. The human 14-3-3gamma gene (HGMW-approved symbol YWHAG) is highly expressed in brain, skeletal muscle, and heart. By fluorescence in situ hybridization analysis, the human 14-3-3gamma gene was mapped to chromosome 7q11.23. Radiation hybrid mapping has shown that this gene is localized 2.33 cR telomeric to D7S1870, a polymorphic marker located at the most telomeric end of the common deletion region of Williams-Beuren syndrome (WBS). This suggests that haploinsufficiency of 14-3-3gamma may not contribute to the WBS phenotype. However, information regarding the precise chromosomal location of a member of the 14-3-3 family of genes will aid in examining the relationship between this family of proteins and human disorders.     

The embryonic expression patterns of zebrafish genes encoding LysM-domains

The function and structure of LysM-domain containing proteins are very diverse. Although some LysM domains are able to bind peptidoglycan or chitin type carbohydrates in bacteria, in fungi and in plants, the function(s) of vertebrate LysM domains and proteins remains largely unknown. In this study we have identified and annotated the six zebrafish genes of this family, which encode at least ten conceptual LysM-domain containing proteins. Two distinct sub-families called LysMD and OXR were identified and shown to be highly conserved across vertebrates. The detailed characterization of LysMD and OXR gene expression in zebrafish embryos showed that all the members of these sub-families are strongly expressed maternally and zygotically from the earliest stages of a vertebrate embryonic development. Moreover, the analysis of the spatio-temporal expression patterns, by whole mount and fluorescent in situ hybridizations, demonstrates pronounced LysMD and OXR gene expression in the zebrafish brain and nervous system during stages of larval development. None of the zebrafish LysMD or OXR genes was responsive to challenge with bacterial pathogens in embryo models of Salmonella and Mycobacterium infections. In addition, the expression patterns of the OXR genes were mapped in a zebrafish brain atlas.     

Comparative analysis of two 14-3-3 homologues and their expression pattern in the root-knot nematode Meloidogyne incognita

14-3-3 proteins are highly conserved ubiquitous proteins found in all eukaryotic organisms. They are involved in various cellular processes including signal transduction, cell-cycle control, apoptosis, stress response and cytoskeleton organisation. We report here the cloning of two genes encoding 14-3-3 isoforms from the plant parasitic root-knot nematode Meloidogyne incognita, together with an analysis of their expression. Both genes were shown to be transcribed in unhatched second stage larvae, infective second stage larvae, adult males and females. The Mi-14-3-3-a gene was shown to be specifically transcribed in the germinal primordium of infective larvae, whereas Mi-14-3-3-b was transcribed in the dorsal oesophageal gland in larvae of this stage. The MI-14-3-3-B protein was identified by mass spectrometry in in vitro-induced stylet secretions from infective larvae. The stability and distribution of MI-14-3-3 proteins in host plant cells was assessed after stable expression of the corresponding genes in tobacco BY2 cells.     

Sequence and expression analysis of a Xenopus laevis cDNA which encodes a homologue of mammalian 14-3-3 zeta protein

We report the cloning and characterisation of a cDNA that encodes a novel member of the Xenopus laevis 14-3-3 protein family. Sequence analysis reveals that the cDNA-encoded protein shares 84% identity with the rat, human or sheep 14-3-3ζ isoform, and between 66% and 77% identity with bovine, human or rat β, bovine γ, human τ, Drosophila 14-3-3 and a previously isolated Xenopus member. The corresponding mRNA is present in all adult tissues examined with the highest levels in the brain. Although the gene is expressed throughout embryogenesis, higher levels of mRNA accumulate after gastrulation. Whole-mount in situ hybridisation on tailbud stage embryo reveals strong expression of the gene in the head, optic vesicles, spinal cord and branchial arches with weaker expression in the somites. In addition, expression along the notochord is observed at stage 45 (tadpole). This spatial and temporal expression profile along with recent studies implicating the importance of 14-3-3 proteins in the regulation of signal transduction pathways argues for a key role of this isoform in embryonic development.     

Unraveling 14-3-3 Proteins in C4 Panicoids with Emphasis on Model Plant Setaria italica Reveals Phosphorylation-Dependent Subcellular Localization of RS Splicing Factor

14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C₄ panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides interesting information on their gene structure, protein domains, phylogenetic and evolutionary relationships, and expression patterns during abiotic stresses and hormonal treatments, which could be useful in choosing candidate members for further functional characterization. In addition, demonstration of interaction between Si14-3-3 and SiRSZ21A provides novel clues on the involvement of 14-3-3 proteins in the splicing events.     

The HMGB protein gene family in zebrafish: Evolution and embryonic expression patterns

The High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes.