Phase-dependent Expression Research Articles

A TrmBL2-like transcription factor mediates the growth phase-dependent expression of halolysin SptA in a concentration-dependent manner in Natrinema gari J7-2.

Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.

Allele-specific DNA methylation and gene expression during shoot organogenesis in tissue culture of hybrid poplar.

Plant tissue regeneration is critical for genetic transformation and genome editing techniques. During the regeneration process, changes in epigenetic modifications accompany the cell fate transition. However, how allele-specific DNA methylation in two haplotypes contributes to the transcriptional dynamics during regeneration remains elusive. Here we applied an inter-species hybrid poplar (Populus alba × P. glandulosa cv. 84K) as a system to characterize the DNA methylation landscape during de novo shoot organogenesis at allele level. Both direct and indirect shoot organogenesis showed a reduction in genome-wide DNA methylation. At gene level, non-expressed genes were hypermethylated in comparison with expressed genes. Among the genes exhibiting significant correlations between levels of DNA methylation and gene expression, the expression patterns of 75% of genes were negatively correlated with DNA methylation in the CG context, whereas the correlation patterns in the CHH context were the reverse. The allele-biased DNA methylation was consistent during shoot organogenesis, with fewer than one-thousandth of allele-specific methylation regions shifted. Analysis of allele-specific expression revealed that there were only 1909 genes showing phase-dependent allele-biased expression in the regeneration process, among which the allele pairs with greater differences in transcription factor binding sites at promoter regions exhibited greater differences in allele expression. Our results indicated a relatively independent transcriptional regulation in two subgenomes during shoot organogenesis, which was contributed by cis-acting genomic and epigenomic variations.

Exploring the possible role of estradiol in the regulation of reproductive phase-dependent expression of NOD-like receptors in female Channa punctata

Exploring the possible role of estradiol in the regulation of reproductive phase-dependent expression of NOD-like receptors in female Channa punctata

Nesfatin-1 in a reptile: its role and hormonal regulation in wall lizard testis

Nesfatin-1 is a pleiotropic hormone implicated in various physiological functions including reproduction. Studies though limited, have established an important role of the peptide in regulation of testicular functions in mammals and fishes. However, role of nesfatin-1 in regulation of spermatogenesis and testicular steroidogenesis remains completely unexplored in reptiles. Therefore, present study aimed to develop an insight into reproductive phase-dependent testicular expression, function and regulation of nucb2/nesfatin-1 in a reptile, Hemidactylus flaviviridis. Expression of nucb2/nesfatin-1 in testis of wall lizard varied significantly depending upon reproductive phase, being highest in the active phase while lowest during regressed phase. Further, in vitro treatment of wall lizard testis with nesfatin-1 showed a concentration- and time-dependent stimulatory effect of the peptide on expression of cell proliferation and differentiation markers like scf, c-kit and pcna suggesting a spermatogenic role of nesfatin-1 in wall lizard. Also, nesfatin-1 stimulated the anti-apoptotic marker, bcl-2 while inhibited the apoptotic marker, caspase-3, suggesting its role as an inhibitor of apoptosis of testicular cells. Further, treatment with nesfatin-1 resulted in significantly higher expression of star along with a concomitant increase in testosterone production by the lizard testis. The present study also demonstrates hormonal regulation of testicular nucb2/nesfatin-1 wherein follicle-stimulating hormone (FSH) inhibited while sex steroids like dihydrotestosterone (DHT) and 17β-estradiol-3-benzoate (E2) stimulated the mRNA expression of nesfatin-1. Observations from the current study for the first time provide comprehensive evidence of spermatogenic and steroidogenic role of nesfatin-1 as well as its hormonal regulation in the testis of a reptile, H. flaviviridis.

The role of replication-induced chromosomal copy numbers in spatio-temporal gene regulation and evolutionary chromosome plasticity.

For a coherent response to environmental changes, bacterial evolution has formed a complex transcriptional regulatory system comprising classical DNA binding proteins sigma factors and modulation of DNA topology. In this study, we investigate replication-induced gene copy numbers - a regulatory concept that is unlike the others not based on modulation of promoter activity but on replication dynamics. We show that a large fraction of genes are predominantly affected by transient copy numbers and identify cellular functions and central pathways governed by this mechanism in Escherichia coli. Furthermore, we show quantitatively that the previously observed spatio-temporal expression pattern between different growth phases mainly emerges from transient chromosomal copy numbers. We extend the analysis to the plant pathogen Dickeya dadantii and the biotechnologically relevant organism Vibrio natriegens. The analysis reveals a connection between growth phase dependent gene expression and evolutionary gene migration in these species. A further extension to the bacterial kingdom indicates that chromosome evolution is governed by growth rate related transient copy numbers.

Bidirectional hybrid erythritol-inducible promoter for synthetic biology in Yarrowia lipolytica

BackgroundThe oleaginous yeast Yarrowia lipolytica is increasingly used as a chassis strain for generating bioproducts. Several hybrid promoters with different strengths have been developed by combining multiple copies of an upstream activating sequence (UAS) associated with a TATA box and a core promoter. These promoters display either constitutive, phase-dependent, or inducible strong expression. However, there remains a lack of bidirectional inducible promoters for co-expressing genes in Y. lipolytica.ResultsThis study built on our previous work isolating and characterizing the UAS of the erythritol-induced genes EYK1 and EYD1 (UAS-eyk1). We found an erythritol-inducible bidirectional promoter (BDP) located in the EYK1-EYL1 intergenic region. We used the BDP to co-produce YFP and RedStarII fluorescent proteins and demonstrated that the promoter’s strength was 2.7 to 3.5-fold stronger in the EYL1 orientation compared to the EYK1 orientation. We developed a hybrid erythritol-inducible bidirectional promoter (HBDP) containing five copies of UAS-eyk1 in both orientations. It led to expression levels 8.6 to 19.2-fold higher than the native bidirectional promoter. While the BDP had a twofold-lower expression level than the strong constitutive TEF promoter, the HBDP had a 5.0-fold higher expression level when oriented toward EYL1 and a 2.4-fold higher expression level when oriented toward EYK1. We identified the optimal media for BDP usage by exploring yeast growth under microbioreactor conditions. Additionally, we constructed novel Golden Gate biobricks and a destination vector for general use.ConclusionsIn this research, we developed novel bidirectional and hybrid bidirectional promoters of which expression can be fine-tuned, responding to the need for versatile promoters in the yeast Y. lipolytica. This study provides effective tools that can be employed to smoothly adjust the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.

Analytical spectrum of nonlinear Thomson scattering including radiation reaction

Accelerated charges emit electromagnetic radiation and the consequent energy-momentum loss alters their trajectory. This phenomenon is known as radiation reaction and the Landau-Lifshitz (LL) equation is the classical equation of motion of the electron, which takes into account radiation-reaction effects in the electron trajectory. By using the analytical solution of the LL equation in an arbitrary plane wave, we compute the analytical expression of the classical emission spectrum via nonlinear Thomson scattering including radiation-reaction effects. Both the angularly resolved and the angularly integrated spectra are reported, which are valid in an arbitrary plane wave. Also, we have obtained a phase-dependent expression of the electron dressed mass, which includes radiation-reaction effects. Finally, the corresponding spectra within the locally constant field approximation have been derived.

Transcriptome-Wide Analysis of Stationary Phase Small ncRNAs in E. coli.

Almost two-thirds of the microbiome’s biomass has been predicted to be in a non-proliferating, and thus dormant, growth state. It is assumed that dormancy goes hand in hand with global downregulation of gene expression. However, it remains largely unknown how bacteria manage to establish this resting phenotype at the molecular level. Recently small non-protein-coding RNAs (sRNAs or ncRNAs) have been suggested to be involved in establishing the non-proliferating state in bacteria. Here, we have deep sequenced the small transcriptome of Escherichia coli in the exponential and stationary phases and analyzed the resulting reads by a novel biocomputational pipeline STARPA (Stable RNA Processing Product Analyzer). Our analysis reveals over 12,000 small transcripts enriched during both growth stages. Differential expression analysis reveals distinct sRNAs enriched in the stationary phase that originate from various genomic regions, including transfer RNA (tRNA) fragments. Furthermore, expression profiling by Northern blot and RT-qPCR analyses confirms the growth phase-dependent expression of several enriched sRNAs. Our study adds to the existing repertoire of bacterial sRNAs and suggests a role for some of these small molecules in establishing and maintaining stationary phase as well as the bacterial stress response. Functional characterization of these detected sRNAs has the potential of unraveling novel regulatory networks central for stationary phase biology.

Phase-dependent expression profiling and quantification of several growth factors in liver regeneration after partial hepatectomy

Growth factors are the potential operational members which control different phases of liverregeneration. Different growth factors have expression regulation in the whole process relating todifferent phases of liver regeneration. Objective: To assess the expression regulation of differentgrowth factors and cytokines involved in liver regeneration in a phase-dependent manner. Methods:Blood and liver samples were collected and analyzed on 1st, 3rd, 5th, 7th and 14th postoperative days after50% Partia hepatectomy (PHx). Results: Steady increase of liver regeneration rate was recorded from90.8% (1st day) to 97.9% (7th day). Liver function tests further confirmed the steady liver recovery in PHxmice. Several growth factors such as HGF and VEGF exhibited an up-regulation till 5th day and latergradual decrease till 14th day compared to control mice. Albumin, CK18 and CK19 showed sequentialexpression increase from 1st to 14th day compared to AFP and HNF-4α upregulated until 5th and 1st day,respectively. Quantification of these growth factors further confirm our results. Conclusions:Conclusively, these results highlight a phase-dependent regulation and role of growth factors in liverregeneration and recovery .

Differential Regulation of CsrC and CsrB by CRP-cAMP in Salmonella enterica.

Post-transcriptional regulation mediated by regulatory small RNAs (sRNAs) has risen as a key player in fine-tuning gene expression in response to environmental stimuli. Here, we show that, in Salmonella enterica, the central metabolic regulator CRP-cAMP differentially regulates the sRNAs CsrB and CsrC in a growth phase-dependent manner. While CsrB expression remains unchanged during growth, CsrC displays a growth phase-dependent expression profile, being weakly expressed at the logarithmic growth phase and induced upon entry into stationary phase. We show that CRP-cAMP contributes to the expression pattern of CsrC by repressing its expression during the logarithmic growth phase. The CRP-cAMP mediated repression of CsrC is independent of SirA, a known transcriptional CsrB/CsrC activator. We further show that the sRNA Spot 42, which is derepressed in a Δcrp strain, upregulates CsrC during logarithmic growth. We propose a model where the growth-dependent regulation of CsrC is sustained by the CRP-cAMP-mediated repression of Spot 42. Together, our data point toward a differential regulation of the sRNAs CsrB and CsrC in response to environmental stimuli, leading to fine-tuning of gene expression via the sequestration of the RNA-binding protein CsrA.

Dendrobium officinale Kimura et Migo and American ginseng mixture: A Chinese herbal formulation for gut microbiota modulation.

Dendrobium officinale Kimura et Migo (D. officinale) is a famous traditional Chinese medicine (TCM). A mixture of D. officinale and American ginseng has been shown to enhance cell-mediated immunity, humoral immunity, and monocyte/macrophage functions in mice. Here, the effects of a D. officinale and American ginseng mixture on the structure of gut microbial community in dogs were examined using high-throughput 16S rRNA gene amplicon sequencing. The data revealed that while the mixture did not change the diversity of gut microbial community significantly, differences among individuals were significantly reduced. Furthermore, the mixture-responsive operational taxonomic units (OTUs) exhibited a phase-dependent expression pattern. Fifty-five OTUs were found to exhibit a mixture-induced expression pattern, among which one third were short-chain fatty acid (SCFA)-producing genera and the others were probiotic genera included Lactobacillus spp., Sutterella, Alistipes, Anaerovorax, Bilophila, Coprococcus, Gordonibacter, Oscillibacter, among others. By contrast, 36% of the OTUs exhibiting a mixture-repressed expression pattern were disease-associated microorganisms, and six genera, namely Actinomyces, Escherichia/Shigella, Fusobacterium, Slackia, Streptococcus and Solobacterium, were associated with cancer. In addition, five genera were closely associated with diabetes, namely Collinsella, Rothia, Howardella, Slackia and Intestinibacter. Our results indicate that this D. officinale and American ginseng mixture may be used as a prebiotic agent to enhance SCFA-producing genera and prevent gut dysbiosis.

Reproductive phase-dependent variation, sexually dimorphic expression and sex steroids-mediated transcriptional regulation of lep and lepr in lymphoid organs of Channa punctata

The reproductive phase-dependent and sex-related differential expression of leptin (lep) and its receptor (lepr) in primary and secondary lymphoid organs of a highly nutritive economically important Channa punctata preempts the involvement of sex steroids in modulating intra-immuno-leptin system. This hypothesis was strengthened when plasma testosterone (T) and estradiol (E2) levels in male and female fish of reproductively active spawning and quiescent phases were correlated with lep and lepr expression in their immune organs. Splenic lep and lepr showed a negative correlation with T in both male and female, while with E2 there was a positive correlation in male and negative in female C. punctata. In head kidney, a contrasting correlation was observed as compared to spleen. To validate the implication of sex steroids in regulating leptin system in immune organs, in vivo and in vitro experiments were performed with DHT and E2. Upon administration, lep and lepr expression in tissues of either sex was downregulated. In addition, in vitro results with either of the sex steroids exemplified their direct involvement. Overall, this study, for the first time, reports correlation between sex steroids and transcript expression of leptin system in immune organs of a seasonally breeding vertebrate.

Sirtuin SirD is involved in α-amylase activity and citric acid production in Aspergillus luchuensis mut. kawachii during a solid-state fermentation process

Sirtuins are understood to play a significant role in growth phase-dependent gene expression. In the study presented here, we examined the sirtuins in the white koji fungus, Aspergillus luchuensis mut. kawachii (Aspergillus kawachii), to examine their role in the regulation of amylolytic enzymes and citric acid production during solid-state culture (koji). Characterization of rice koji made using five sirtuin gene disruptants indicated that these genes are involved in the amylolytic activity and acidity of rice koji; the sirD disruptant in particular showed lower levels of acid-stable α-amylase activity and citric acid production per mycelial weight in koji compared to the control strain. The sirD disruptant also showed a change in mycelial pigmentation, and had higher sensitivity to cell wall biogenesis inhibitors such as calcofluor white and Congo red and reduced conidia formation. These results indicate that SirD is required for secondary metabolism, cell wall integrity, and conidial development. Cap analysis gene expression (CAGE) and quantitative RT-PCR analysis indicated that transcriptional changes were related to the characteristic phenotype of the sirD disruptant, including a reduced transcript level of the acid-stable α-amylase gene and a citric acid exporter in rice koji. These results indicate that SirD has a significant role in global transcriptional regulation, including the production of acid-stable α-amylase and citric acid, in A.kawachii during the solid-state fermentation process.

In silico analysis, seasonal variation and gonadotropic regulation of jag1 and its receptor notch1 in testis of spotted snakehead Channa punctatus

The present study in seasonally breeding spotted snakehead Channa punctatus, for the first time in nonmammalian vertebrates, demonstrated correlation between reproductive phase-dependent testicular expression of ligand Jag1/receptor Notch1 and spermatogenic events. Testicular transcriptome sequencing data from our earlier study in C. punctatus was used in the present study to select the best transcript for jag1 (cpjag1) and notch1 (cpnotch1). The transcripts cpjag1 and cpnotch1 encoded full-length putative proteins of 1215 (cpJag1) and 2475 (cpNotch1) amino acids, respectively. A marked homology in the extracellular domains of Jag1 and Notch1 was observed following their alignment with respective proteins from different vertebrates, suggesting conservation in ligand-receptor interaction in C. punctatus. Both cpJag1 and cpNotch1 showed phylogenetic closeness with their teleostean counterparts, especially with that of Perciformes. Temporal expression of cpjag1 and cpnotch1 in testis depending on reproductive phases showed an appreciably high expression during spermatogenically inactive resting and postspawning phases when seminiferous lobules consisted of spermatogonial stem cells and undifferentiated spermatogonia. Their expression sharply declined during spermatogenically active preparatory and spawning phases. It appears that involvement of cpjag1/cpnotch1 is restricted to inactive phases when spermatogonial stem cells renew themselves and replenish undifferentiated spermatogonia. This assumption is ascertained by an experimental study in which high level of testicular cpjag1/cpnotch1 expression in control fish of resting phase markedly decreased after administration of human chorionic gonadotropin that is known to induce proliferation and differentiation of spermatogonia and spawning of spermatozoa.

Small nucleolar RNAS in OA pathogenesis and cartilage biology

Purpose: Small nucleolar RNAs (snoRNAs) are non-coding RNAs of 50-150 nucleotides long with a main task in post-transcriptional modification of ribosomal RNAs (rRNAs). SnoRNAs can be classified into box C/D and box H/ACA snoRNAs. Guided by sequence complementarity between dedicated snoRNAs and positions on the rRNA sequence, the rRNA is site-specifically modified. Box C/D snoRNAs are involved in 2’O-ribose methylation, while box H/ACA in pseudouridylation. A small subset of snoRNAs (e.g., U3, U8 and RMRP), have essential functions in endoribonucleolytic processing of 47S pre-rRNA. Although hundreds of snoRNAs have been identified, their significance is unknown for the cartilage/OA field. We are currently undertaking a number of targeted and discovery studies to investigate the relevance of snoRNAs for chondrocyte differentiation, OA disease progression and OA biomarker identification. Methods: General chondrocyte molecular biology methods were used to isolate, culture and analyse chondrocytes, cartilage and serum/synovial fluid. Transcriptomics was performed using micro-array and RNAseq approaches. Intervention with snoRNA expression was performed by transfecting anti-sense oligonucleotides. Results: Expression of RMRP snoRNA is regulated during chondrogenic differentiation and under control of PTHrP, TGF-beta and BMP-2. RMRP snoRNA function was found to be associated with 47S pre-rRNA processing and necessary for chondrogenic differentiation. Expression of rRNAs and ribosome translation capacity was found to adapt as function of chondrogenic differentiation and RNAseq expression profiling of ATDC5 chondrogenic differentiation uncovered a variety of snoRNAs with differentiation phase-dependent expression profiles. We investigated snoRNA expression in mouse knee joint ageing and post-traumatic OA (DMM). Ageing and OA development led to alterations of specific snoRNA expression in whole mouse joints. Micro-array expression profiling of young, old-protected and old-OA human articular knee cartilage demonstrated age-specific and OA-specific expression of snoRNAs. We are now interrogating whether age- and OA-specific snoRNAs functionally interact with the chondrocyte phenotype and may relate to OA-associated changes in chondrocyte biology. Indeed we have found evidence that alterations in U3 snoRNA expression leads to OA-like changes in the chondrocyte phenotype and that expression of U3 snoRNA can be influenced by morphogens and cytokines. This is associated with changes in 47S pre-rRNA maturation and mature rRNA levels. Also knock-down of OA-specific snoRNAs SNORD78, SNORD26 or SNORD96 induces changes in chondrocyte differentiation status and rRNA levels. Our snoRNA biomarker studies have identified ageing- and OA-specific expression of snoRNAs. In a mouse ageing model we discovered that in the serum of aged mice higher expression of SNORA31 and SNORD23 can be detected and that SNORD116 is dramatically elevated in the serum of mouse and equine OA subjects. We are now exploring the snoRNA content of healthy and OA synovial fluid to identify snoRNA biomarkers for early OA development and investigate whether synovial fluid-mediated inter-tissue communication within the joint involves snoRNAs. Conclusions: Collectively our data show for the first time that specific snoRNAs are differentially expressed due to chondrocyte differentiation and in ageing- and OA cartilage. Expression of specific snoRNAs responds to morphogens and cytokines and our identified snoRNAs influence the phenotypic behaviour of the chondrocyte, potentially via alterations in processing and post-trancriptional modification of rRNAs. SnoRNAs provide an unexplored class of non-coding RNAs relevant for chondrocyte biology. We are now studying further in-depth the variety of identified snoRNAs in chondrocyte differentiation, -homeostasis and -disease processes and identifying snoRNA candidates that may be used as potential biomarkers for OA.

Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1 Streptococcus pyogenes

Streptococcus pyogenes (group A Streptococcus [GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation of speB expression is linked to disease severity phenotypes. Here, we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1 serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and Western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction of speB gene expression. Compared to wild-type and complemented mutant strains, we demonstrate that the 5448ΔpepO mutant strain is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections.IMPORTANCE Despite the continuing susceptibility of S. pyogenes to penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and noninvasive diseases in Western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on the regulatory mechanisms that control speB gene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator of speB gene expression in the globally disseminated M1T1 clone and as being essential for virulence.

Three tandem promoters, together with IHF, regulate growth phase dependent expression of the Escherichia coli kps capsule gene cluster

In this study we characterise three tandem promoters (PR1-1, PR1-2 and PR1-3) within the PR1 regulatory region of the Escherichia coli kps capsule gene cluster. Transcription from promoter PR1-2 was dependent on the activity of the upstream promoter PR1-1, which activated PR1-2 via transcription coupled DNA supercoiling. During growth at 37 °C a temporal pattern of transcription from all three promoters was observed with maximum transcriptional activity evident during mid-exponential phase followed by a sharp decrease in activity as the cells enter stationary phase. The growth phase dependent transcription was regulated by Integration Host Factor (IHF), which bound within the PR1 region to repress transcription from PR1-2 and PR1-3. This pattern of transcription was mirrored by growth phase dependent expression of the K1 capsule. Overall these data reveal a complex pattern of transcriptional regulation for an important virulence factor with IHF playing a role in regulating growth phase expression.

Temporal expression and gonadotropic regulation of aromatase and estrogen receptors in the ovary of wall lizard, Hemidactylus flaviviridis: Correlation with plasma estradiol and ovarian follicular development

The current study in Indian wall lizard Hemidactylus flaviviridis for the first time demonstrates the reproductive phase-dependent expression pattern of aromatase (cyp19) and estrogen receptor subtypes (er-α and er-β) as well as their gonadotropic regulation in the ovary of a squamate. The expression of cyp19 remained low during regressed phase, increased markedly in recrudescent and declined sharply in breeding phase. Further, temporal profile of plasma estradiol 17-β (E2) was found to be relatively parallel to the expression pattern of ovarian cyp19. The expression pattern of estrogen receptors in the ovary showed subtype-specific variation along the reproductive cycle. Expression of ovarian er-α remained high from regressed to late recrudescence, while er-β expression that was low during regression dramatically increased with the initiation of follicular growth in early recrudescence and remained high until late recrudescence. Nonetheless, expression of both the receptors declined during breeding phase when ovary contained vitellogenic follicle. Regarding gonadotropic regulation, short-term treatment with Follicle stimulating hormone (3 injections of FSH) increased the ovarian expression of cyp19, er-α and er-β while prolongation of treatment (7 or 11 injections) resulted in a marked decrease in expression of these genes concomitant to formation of vitellogenic follicle. However, a marked increase in plasma E2 was recorded after 7 injections of FSH. The direct role of gonadotropin in regulation of cyp19 and estrogen receptors was established by an in vitro study where FSH upregulated the expression of these genes in all stages of ovarian follicles (early growing, previtellogenic and early vitellogenic) of wall lizards.

Molecular dissection of transcriptional reprogramming of steviol glycosides synthesis in leaf tissue during developmental phase transitions in Stevia rebaudiana Bert

Stevia is a natural source of commercially important steviol glycosides (SGs), which share biosynthesis route with gibberellic acids (GAs) through plastidal MEP and cytosolic MVA pathways. Ontogeny-dependent deviation in SGs biosynthesis is one of the key factor for global cultivation of Stevia, has not been studied at transcriptional level. To dissect underlying molecular mechanism, we followed a global transcriptome sequencing approach and generated more than 100 million reads. Annotation of 41,262 de novo assembled transcripts identified all the genes required for SGs and GAs biosynthesis. Differential gene expression and quantitative analysis of important pathway genes (DXS, HMGR, KA13H) and gene regulators (WRKY, MYB, NAC TFs) indicated developmental phase dependent utilization of metabolic flux between SGs and GAs synthesis. Further, identification of 124 CYPs and 45 UGTs enrich the genomic resources, and their PPI network analysis with SGs/GAs biosynthesis proteins identifies putative candidates involved in metabolic changes, as supported by their developmental phase-dependent expression. These putative targets can expedite molecular breeding and genetic engineering efforts to enhance SGs content, biomass and yield. Futuristically, the generated dataset will be a useful resource for development of functional molecular markers for diversity characterization, genome mapping and evolutionary studies in Stevia.

Growth phase-dependent expression profiles of three vital H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440 and on the pCAR1 plasmid

BackgroundH-NS family proteins are nucleoid-associated proteins that form oligomers on DNA and function as global regulators. They are found in both bacterial chromosomes and plasmids, and were suggested to be candidate effectors of the interaction between them. TurA and TurB are the predominantly expressed H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440, while Pmr is encoded on the carbazole-degradative incompatibility group P-7 plasmid pCAR1. Previous transcriptome analyses suggested that they function cooperatively, but play different roles in the global transcriptional network. In addition to differences in protein interaction and DNA-binding functions, cell expression levels are important in clarifying the detailed underlying mechanisms. Here, we determined the precise protein amounts of TurA, TurB, and Pmr in KT2440 in the presence and absence of pCAR1.ResultsThe intracellular amounts of TurA and TurB in KT2440 and KT2440(pCAR1) were determined by quantitative western blot analysis using specific antibodies. The amount of TurA decreased from the log phase (~80,000 monomers per cell) to the stationary phase (~20,000 monomers per cell), while TurB was only detectable upon entry into the stationary phase (maximum 6000 monomers per cell). Protein amounts were not affected by pCAR1 carriage. KT2440(pCAR1pmrHis), where histidine-tagged Pmr is expressed under its original promotor, was used to determine the intracellular amount of Pmr, which was constant (~30,000 monomers per cell) during cell growth. Quantitative reverse transcription PCR demonstrated that the transcriptional levels of turA and turB were consistent with protein expression, though the transcriptional and translational profiles of Pmr differed.ConclusionThe amount of TurB increases as TurA decreases, and the amount of Pmr does not affect the amounts of TurA and TurB. This is consistent with our previous observation that TurA and TurB play complementary roles, whereas Pmr works relatively independently. This study provides insight into the molecular mechanisms underlying reconstitution of the transcriptional network in KT2440 by pCAR1 carriage.