Analysis Of Temporal Expression Patterns Research Articles

Co-expression networks regulating cotton fiber initiation generated by comparative transcriptome analysis between fiberless XZ142FLM and GhVIN1i

Cotton produces natural fiber as crucial raw material for textile industry and thus is economically one of the most important crops worldwide. Currently the mechanisms that control fiber initiation on ovules are still not well understood. In this study, we performed comparative transcriptome analysis between the natural fiberless mutant XZ142FLM and transgenic fiberless line GhVIN1i generated by RNAi silencing of GhVIN1 to identify molecular regulatory networks controlling fiber initiation. It was found that XZ142FLM and GhVIN1i shared many common differentially expressed genes (DEGs) in ovules during fiber initiation, and their respective DEGs were enriched in several identical pathways related to fiber initiation. Many reported fiber initiation or elongation related genes including GhMML3, GhVIN1, GhMYB25, GhHD-1, GhHOX3 exhibited similar expression patterns in XZ142FLM and GhVIN1i during fiber initiation, suggesting similar mechanisms of fiber initiation operating in the two fiberless lines, which was further verified by comparative temporal expression pattern analysis between TM-1/XZ142FLM and Coker 312/GhVIN1i. Co-expression regulatory networks for controlling fiber initiation were constructed for XZ142FLM and GhVIN1i, respectively, which centered on GhMML3 or GhVIN1 with many known fiber initiation/elongation related genes. Several transcription factors and hub genes were identified in the co-expression networks generated, which may facilitate future research in identification of novel candidate genes regulating fiber initiation and elongation. Overall, the results obtained in this study shed light on the regulatory networks mediated by GhMML3 and GhVIN1 for controlling fiber initiation in cotton.

Comparative transcriptome analysis provides insights into grain filling commonalities and differences between foxtail millet [Setaria italica (L.) P. Beauv.] varieties with different panicle types.

Grain filling affects grain weight and quality and is among the most critical factors in determining the yield and quality of cereal crops. Though hybrids have larger panicles and numerous spikelets with a larger sink capacity than conventional varieties, data on the grain filling commonalities and differences between foxtail millet varieties with different panicle types remain sparse. In this study, we found that “Zhang Gu 13” (ZG, large panicle) exhibits a significantly higher panicle weight than “Yu Gu 18” (YG, conventional panicle) at the early stage of grain filling, but the weight of YG increased rapidly and gradually overtook ZG during the middle stages. A temporal expression pattern analysis demonstrated that the genes involved in photosynthesis, metabolic pathways, and phenylpropanoid biosynthesis were downregulated, while those related to peroxisome function, purine metabolism, and zeatin biosynthesis were upregulated during grain filling in both varieties. A total of 6,832 differentially expressed genes (DEGs) were identified in both varieties, with the majority identified at the early and late stages. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further revealed that the upregulated DEGs in YG were associated with gibberellin (GA) biosynthesis, ATP-binding cassette (ABC) transporters, and plant hormone signal transduction. Photosynthesis-related DEGs, such as photosystem and antenna proteins, were significantly upregulated in ZG. This study provides preliminary insights into the differences in gene expression and molecular mechanisms of grain filling between ZG and YG in the North China summer-sowing region.

Heterologous expression and functional characterization of the ligand-binding domain of oxysterol-binding protein from Aspergillus oryzae.

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (β-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.

Integrated analysis of mRNA-seq and miRNA-seq for host susceptibilities to influenza A (H7N9) infection in inbred mouse lines.

Host genetic factors play an important role in diverse host outcomes after influenza A (H7N9) infection. Studying differential responses of inbred mouse lines with distinct genetic backgrounds to influenza virus infection could substantially increase our understanding of the contributory roles of host genetic factors to disease severity. Here, we utilized an integrated approach of mRNA-seq and miRNA-seq to investigate the transcriptome expression and regulation of host genes in C57BL/6J and DBA/2J mouse strains during influenza virus infection. The differential pathogenicity of influenza virus in C57BL/6J and DBA/2J has been fully demonstrated through immunohistochemical staining, histopathological analyses, and viral replication assessment. A transcriptional molecular signature correlates to differential host response to infection has been uncovered. With the introduction of temporal expression pattern analysis, we demonstrated that host factors responsible for influenza virus replication and host-virus interaction were significantly enriched in genes exhibiting distinct temporal dynamics between different inbred mouse lines. A combination of time-series expression analysis and temporal expression pattern analysis has provided a list of promising candidate genes for future studies. An integrated miRNA regulatory network from both mRNA-seq and miRNA-seq revealed several regulatory modules responsible for regulating host susceptibilities and disease severity. Overall, a comprehensive framework for analyzing host susceptibilities to influenza infection was established by integrating mRNA-seq and miRNA-seq data of inbred mouse lines. This work suggests novel putative molecular targets for therapeutic interventions in seasonal and pandemic influenza.

Tumor Evolution Inferred by Patterns of microRNA Expression through the Course of Disease, Therapy, and Recurrence in Breast Cancer.

Molecular evolution of tumors during progression, therapy, and metastasis is a major clinical challenge and the main reason for resistance to therapy. We hypothesized that microRNAs (miRNAs) that exhibit similar variation of expression through the course of disease in several patients have a significant function in the tumorigenic process. Exploration of evolving disease by profiling 800 miRNA expression from serial samples of individual breast cancer patients at several time points: pretreatment, posttreatment, lymph nodes, and recurrence sites when available (58 unique samples from 19 patients). Using a dynamic approach for analysis, we identified expression modulation patterns and classified varying miRNAs into one of the eight possible temporal expression patterns. The various patterns were found to be associated with different tumorigenic pathways. The dominant pattern identified an miRNA set that significantly differentiated between disease stages, and its pattern in each patient was also associated with response to therapy. These miRNAs were related to tumor proliferation and to the cell-cycle pathway, and their mRNA targets showed anticorrelated expression. Interestingly, the level of these miRNAs was lowest in matched recurrent samples from distant metastasis, indicating a gradual increase in proliferative potential through the course of disease. Finally, the average expression level of these miRNAs in the pretreatment biopsy was significantly different comparing patients experiencing recurrence to recurrence-free patients. Serial tumor sampling combined with analysis of temporal expression patterns enabled to pinpoint significant signatures characterizing breast cancer progression, associated with response to therapy and with risk of recurrence. Clin Cancer Res; 22(14); 3651-62. ©2016 AACR.

Multiprotein bridging factor 2 regulates the expression of the fibroin heavy chain gene by interacting with Bmdimmed in the silkworm Bombyx mori.

Multiprotein bridging factor 2 (MBF2) was first isolated from the posterior silk gland of Bombyx mori. However, its function in B.mori is still unknown. Herein, MBF2 transcripts were detected mainly in the posterior silk gland and Malpighian tubules of B.mori larvae via a quantitative PCR analysis. An analysis of temporal expression patterns showed that the expression pattern of MBF2 was the opposite of that of the fibroin heavy chain (fibH) gene, as its expression was high during the fourth-instar moulting stage, decreased gradually during the fifth-instar feeding stage and disappeared at the end of the fifth-instar phase. Furthermore, bimolecular fluorescent complementation and Far-Western blot assays showed that MBF2 interacted with the basic helix-loop-helix transcription factor Bmdimmed. Dual luciferase reporter assays showed that MBF2 down-regulated the promoter activity of fibH and inhibited the effect of Bmdimmed (Bmdimm) on fibH expression. MBF2 expression was induced in silk glands after treatment with 20-hydroxyecdysone in vivo and in vitro. These findings suggest that MBF2 is a transcriptional repressor that is involved in controlling the regulation of the fibH gene in the posterior silk gland by interacting with Bmdimm.

Virion-associated viral proteins of a Chinese giant salamander (Andrias davidianus) iridovirus (genus Ranavirus) and functional study of the major capsid protein (MCP)

Chinese giant salamander iridovirus (CGSIV) is the emerging causative agent to farmed Chinese giant salamanders in nationwide China. CGSIV is a member of the common midwife toad ranavirus (CMTV) subset of the amphibian-like ranavirus (ALRV) in the genus Ranavirus of Iridoviridae family. However, viral protein information on ALRV is lacking. In this first proteomic analysis of ALRV, 40 CGSIV viral proteins were detected from purified virus particles by liquid chromatography-tandem mass spectrometry analysis. The transcription products of all 40 identified virion proteins were confirmed by reverse transcription polymerase chain reaction analysis. Temporal expression pattern analysis combined with drug inhibition assay indicated that 37 transcripts of the 40 virion protein genes could be classified into three temporal kinetic classes, namely, 5 immediate early, 12 delayed early, and 20 late genes. The presence of major capsid proteins (MCP, ORF019L) and a proliferating cell nuclear antigen (ORF025L) was further confirmed by Western blot analysis. The functions of MCP were also determined by small interfering RNA (siRNA)-based knockdown assay and anti-recombinant MCP serum-based neutralization testing. At low dosages of CGSIV, siRNA-based knockdown of the MCP gene effectively inhibited CGSIV replication in fathead minnow cells. The antiviral effect observed in the anti-MCP serum-based neutralization test confirms the crucial function of the MCP gene in CGSIV replication. Taken together, detailed information on the virion-associated viral proteins of ALRV is presented for the first time. Our results also provide evidence that MCP is essential for CGSIV replication in vitro.

Identification and characterization of a novel capsid protein encoded by Singapore grouper iridovirus ORF038L

Singapore grouper iridovirus (SGIV) is an important pathogen isolated from grouper, Epinephelus tauvina, and characterized as a novel ranavirus. To better understand the function of viral structural genes involved in SGIV infection and virus-host interactions, a candidate gene, VP38 (ORF038L), was investigated in this study. SGIV VP38 was found to encode a 170-aa peptide containing an RGD motif, and it showed significant identity only to members of the genus Iridovirus, family Iridoviridae, except megalocytivirus. The VP38 gene was identified by temporal expression pattern analysis and drug inhibition assay as a late (L) gene. Immunofluorescence localization revealed that P38 was distributed predominately in the cytoplasm and that association of VP38 with viral factories increased at the late stage of SGIV infection. Consistent results from immunoelectron microscopy (IEM) and western blot analysis revealed that SGIV VP38 is a viral capsid protein. Furthermore, antibodies specific for SGIV VP38 exhibited substantial SGIV-neutralizing activity in vitro, suggesting that VP38 might play an important role in SGIV infectivity.

Cloning and blood‐meal dependent induction pattern of apolipophorin‐III from Anopheles sinensis

AbstractApolipophorin‐III is known to play a role in transporting lipids in insects, and much attention has been paid to lepidopteran insects' apolipophorin. Thus, we were interested in examining the effects of blood‐meal on the expression pattern of apolipophorin‐III in mosquitoes. This led us to clone and partially characterize the full‐length cDNA of apoLp‐III (AnsiApoLp‐III) from Anopheles sinensis. Analysis of AnsiApoLp‐III cDNA shows that the 728‐bp sequence has a 582‐bp protein‐coding region with 94 bp of putative 5′ untranslated region and 152 bp of 3′ untranslated region. The deduced amino acid sequence begins with a methionine codon at position 95 and extends to position 674, encompassing a polypeptide of 193 amino acids. AnsiApoLp‐III has the highest identity (63%) to Culex quinquefasciatus apoLp‐III. Temporal expression pattern analysis shows that although AnsiApoLp‐III was expressed at all developmental stages, it was highly detected at egg and adult stages in the female mosquitoes. In addition, we found out that AnsiApoLp‐III was induced in An. sinensis adult females after uptaking a blood‐meal. Spatial expression patterns of AnsiApoLp‐III shows that AnsiApoLp‐III mRNA was strongly induced at day 1 and gradually decreased from day 1 to day 4 in the ovaries. Most interestingly, AnsiApoLp‐III mRNA in the Malpighian tubule was strongly induced at day 1, decreased during days 1–3, and then became elevated again at day 4. These data suggest that blood‐meal influences AnsiApoLp‐III mRNA induction in ovaries and Malpighian tubules. It remains to further elucidate the biological roles of AnsiApoLp‐III in these organs.

Molecular cloning and expression pattern of 14‐3‐3ζ from the malaria vector, Anopheles sinensis

Abstract14‐3‐3 proteins are known to play a pivotal role in cell survival, apoptosis and signal transduction. The 14‐3‐3ζ isoform has been cloned and characterized from many eukaryotic organisms, including the fruit fly and silkworm. However, no study on mosquito 14‐3‐3 has been reported to date. In an attempt to investigate the function of 14‐3‐3 in midgut epithelial cells undergoing apoptosis, a cDNA library was generated from the malaria vector, Anopheles sinensis, which was treated with apoptosis‐inducing Actinomycin‐D. We were able to identify and obtain A. sinensis 14‐3‐3ζ cDNA (Ansi14‐3‐3ζ) from expressed sequence tags (EST) analysis after conducting massive sequencing of the A. sinensis cDNA library. Ansi14‐3‐3ζ has very high homology to 14‐3‐3 homologs of various insects, such as Anopheles gambiae (100%), Aedes aegypti (100%), Drosophila melanogaster (96%), Bombyx mori (93%), Apis mellifera (93%) and Mus musculus (81%), indicating that mosquito 14‐3‐3ζ is a highly conserved gene in diverse organisms. Analysis of temporal expression patterns showed that Ansi14‐3‐3ζ mRNA is highly expressed in egg, early pupae and adult stages and is also expressed, although at low levels, in fourth instar larvae and late pupae. In response to two immune elicitors (lipopolysaccharide and laminarin), no striking induction of 14‐3‐3ζ mRNA was observed in A. sinensis. Further studies of the precise biological function, inducibility and subcellular distribution of 14‐3‐3ζ are required in Plasmodium invasion‐induced apoptotic midgut cells in A. sinensis in the context of the Time Bomb model.

A comparative analysis of green fluorescent protein and β-glucuronidase protein-encoding genes as a reporter system for studying the temporal expression profiles of promoters

The assessment of activity of promoters has been greatly facilitated by the use of reporter genes. However, the activity as assessed by reporter gene is a reflection of not only promoter strength, but also that of the stability of the mRNA and the protein encoded by the reporter gene. While a stable reporter gene product is an advantage in analysing activities of weak promoters, it becomes a major limitation for understanding temporal expression patterns of a promoter, as the reporter product persists even after the activity of the promoter ceases. In the present study we undertook a comparative analysis of two reporter genes, beta-glucuronidase (gus) and green fluorescent protein (sgfp), for studying the temporal expression pattern of tapetum-specific promoters A9 (Arabidopsis thaliana) and TA29 (Nicotiana tabacum). The activity of A9 and TA29 promoters as assessed by transcript profiles of the reporter genes (gus or sgfp ) remained the same irrespective of the reporter gene used. However, while the deduced promoter activity using gus was extended temporally beyond the actual activity of the promoter, sgfp as recorded through its fluorescence correlated better with the transcription profile. Our results thus demonstrate that sgfp is a better reporter gene compared to gus for assessment of temporal activity of promoters. Although several earlier reports have commented on the possible errors in deducing temporal activities of promoters using GUS as a reporter protein, we experimentally demonstrate the advantage of using reporter genes such as gfp for analysis of temporal expression patterns.

Transcriptional differences between the male‐sterile mutant bcms and wild‐type Brassica campestris ssp. chinensis reveal genes related to pollen development

A novel male-sterile mutant which lacks mature pollen, Brassisa campestris male sterile (bcms), was identified in Brassica campestris L. ssp. chinensis Makino (syn. B. rapa ssp. chinensis). Genetic analysis revealed that bcms was controlled by a single recessive mutation locus. Genome-wide transcriptional profiling was performed on the flower buds of both the bcms mutant and the wild-type from which it originated, and profiling analysis indicated that there were numerous changes in gene expression attributable to the gene mutation. This mutation resulted in down-regulation of a variety of genes and up-regulated expression of a few other genes. A total of 51 transcript-derived fragments (TDFs) were isolated: 32 specifically and 12 predominantly accumulated in wild-type flower buds, and two specifically and five predominantly accumulated in bcms flower buds. Sequence analysis showed that some of these TDFs share significant similarities with genes involved in different aspects of cellular development, such as signal transduction, cell wall biosynthesis and regulation. Most other TDFs showed no or very poor sequence similarities to entries in any database and might represent new candidate proteins involved in pollen development. Furthermore, spatial and temporal expression pattern analysis of 20 genes derived from cDNA-amplified fragment length polymorphism in different tissues of both the bcms and wild-type plants revealed their complex and dynamic expression patterns. The bcms mutant and the genes isolated in this paper provide excellent material for future studies on the molecular mechanism of male sterility.

Two alloalleles of Xenopus laevis hairy2 gene—evolution of duplicated gene function from a developmental perspective

Gene duplication is a fundamental source of a new gene in the process of evolution. A duplicated gene is able to accept many kinds of mutations that could lead to loss of function or novel phenotypic diversity. Alternatively, the duplicated genes complementarily lose part of their functions to play original roles as a set of genes, a process called subfunctionalization. Pseudotetraploid frog Xenopus laevis has four sets of genes, and it is generally thought that the alloalleles in X. laevis have mutually indistinguishable functions. In this paper, we report differences and similarities between Xhairy2a and Xhairy2b in the neural crest, floor plate, and prechordal plate. Knockdown studies showed that Xhairy2a seems not to function in the neural crest, although both of them are required in the floor plate and the prechordal plate. Temporal expression pattern analysis revealed that Xhairy2a is a maternal factor having lower zygotic expression than Xhairy2b, while Xhairy2b is not loaded in the egg but has high zygotic expression. Spatial expression pattern analysis demonstrated that future floor plate expression is shared by both alloalleles, but Xhairy2b expression in the neural crest is much higher than Xhairy2a expression, consistent with the results of individual knockdown experiments. Therefore, our data suggest that subfunctionalization occurs in Xhairy2.

THE GH-IGF AXIS AND ITS POTENTIAL ROLE IN THE OVARY OF ZEBRAFISH, Danio rerio

Growth hormone (GH) from the pituitary is the major hormone responsible for body growth in vertebrates. However, there has been increasing evidence that GH is also involved in several reproductive functions including steroidogenesis and gametogenesis in the gonads. In view of this, GH has also been termed co-gonadotropin. It is well established that most effects of GH are mediated by insulin-like growth factors (IGFs) produced in different tissues. In the ovary, however, both IGF-dependent and independent mechanisms have been proposed for GH actions, and one of the factors that work downstream of GH signaling appears to be activin. Interestingly, the expression of GH itself has been detected in the ovary, suggesting the existence of an intraovarian GH-IGF axis. Using zebrafish as the model, the present project was undertaken to characterize the mini-GH-IGF-I axis in the ovary with particular reference to its relationship with the local activin-follistatin system. RTPCR analysis showed that Gh (gh), Igf1 (igf1), Igf2 (igf2), and their receptors were all expressed in the ovary, indicating that a potential intraovarian GH-IGF system also exists in the zebrafish. Further experiment on spatial distribution using isolated follicle layers and denuded oocytes revealed that gh was exclusively expressed in the fullgrown oocytes, while its receptor Ghr (ghr) could be detected in both oocytes and follicle cells. Igf1 and its receptor igf1r as well as igf2 could also be detected in both oocytes and follicle cells. Analysis of temporal expression patterns demonstrated that gh and ghr were differentially expressed during folliculogenesis. The expression level of gh in the follicles seemed to be the highest at the primary growth (PG) stage, but it gradually decreased when PG follicles developed to full-grown (FG) follicles. On the contrary, the expression of ghr was low at the PG stage but significantly increased in the previtellogenic (PV) stage and remained high in later stages. Similarly, the expression of igf1r gradually increased from PG to mid-vitellogenic (MV) stage, but decreased significantly in the final FG stage. Igf1 expression increased from PG to EV stage and decreased afterwards till FG stage. While expression of igf2 maintained constant during folliculogenesis. When tested in cultured follicle cells, recombinant human Igf1 increased the expression of activin beta A (inhba) but decreased that of activin beta B (inhbb) in a clear time and dose-dependent manner. GH (bream and human GH), however, seemed to increase the expression of both inhba and inhbb, which supports our previous observation that GH-induced zebrafish oocyte maturation could be blocked by follistatin, an activin-binding protein. Although we have not obtained convincing evidence for IGFs as the downstream mediators of GH in the zebrafish ovary, our preliminary data did suggest a potential role for the local ovarian activin system in GH as well as IGF signaling. (platform)

Prostaglandin signaling and ovarian follicle development in the silkmoth, Bombyx mori

Previous work on in vitro culturing of silkmoth ( Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.

Induction of jasmonate biosynthesis in arbuscular mycorrhizal barley roots.

Colonization of barley (Hordeum vulgare cv Salome) roots by an arbuscular mycorrhizal fungus, Glomus intraradices Schenck & Smith, leads to elevated levels of endogenous jasmonic acid (JA) and its amino acid conjugate JA-isoleucine, whereas the level of the JA precursor, oxophytodienoic acid, remains constant. The rise in jasmonates is accompanied by the expression of genes coding for an enzyme of JA biosynthesis (allene oxide synthase) and of a jasmonate-induced protein (JIP23). In situ hybridization and immunocytochemical analysis revealed that expression of these genes occurred cell specifically within arbuscule-containing root cortex cells. The concomitant gene expression indicates that jasmonates are generated and act within arbuscule-containing cells. By use of a near-synchronous mycorrhization, analysis of temporal expression patterns showed the occurrence of transcript accumulation 4 to 6 d after the appearance of the first arbuscules. This suggests that the endogenous rise in jasmonates might be related to the fully established symbiosis rather than to the recognition of interacting partners or to the onset of interaction. Because the plant supplies the fungus with carbohydrates, a model is proposed in which the induction of JA biosynthesis in colonized roots is linked to the stronger sink function of mycorrhizal roots compared with nonmycorrhizal roots.