Non-responsive Genes Research Articles

Novel mRNA Signature for Anti-TNF-α Therapy Primary Response in Patients With Ulcerative Colitis.

Ulcerative colitis (UC), an idiopathic, chronic inflammatory disorder of the colonic mucosa, is commonly treated with antitumor necrosis factor α (anti-TNF-α) agents. However, only approximately two-thirds have an initial response to these therapies. We integrated gene expression profiling from 3 independent data sets of 79 UC patients before they began anti-TNF-α therapy and calculated the differentially expressed genes between patient response and nonresponse to anti-TNF-α therapy and developed a de novo response-associated transcription signature score (logOR_Score) to demonstrate the predictive capability of anti-TNF-α therapy for therapeutic efficacy. Furthermore, we performed association analysis of the logOR_Score and clinical features, such as disease activity and immune microenvironment. A total of 2522 responsive and 1824 nonresponsive genes were identified from the integrated data set. Responsive genes were significantly enriched in metabolism-related pathways, whereas nonresponsive ones were associated with immune response-related pathways. The logOR_Score enabled the accurate prediction of the therapeutic efficacy of anti-TNF-α in 4 independent patient cohorts and outperformed the predictions made based on 6 transcriptome-based signatures. In terms of clinical features, the logOR_Score correlated highly with the activity of UC. From an immune microenvironment perspective, logOR_Scores of CD8+IL-17+ T cells, follicular B cells, and innate lymphoid cells significantly decreased in inflamed UC tissue. The de novo response-associated transcription signature may provide novel insights into the personalized treatment of patients with UC. Comprehensive analyses of the response-related subtypes and the association between logOR_Score and clinical features and immune microenvironment may provide insights into the underlying UC pathogenesis.

Temporal regulation of cold transcriptional response in switchgrass.

Switchgrass low-land ecotypes have significantly higher biomass but lower cold tolerance compared to up-land ecotypes. Understanding the molecular mechanisms underlying cold response, including the ones at transcriptional level, can contribute to improving tolerance of high-yield switchgrass under chilling and freezing environmental conditions. Here, by analyzing an existing switchgrass transcriptome dataset, the temporal cis-regulatory basis of switchgrass transcriptional response to cold is dissected computationally. We found that the number of cold-responsive genes and enriched Gene Ontology terms increased as duration of cold treatment increased from 30 min to 24 hours, suggesting an amplified response/cascading effect in cold-responsive gene expression. To identify genomic sequences likely important for regulating cold response, machine learning models predictive of cold response were established using k-mer sequences enriched in the genic and flanking regions of cold-responsive genes but not non-responsive genes. These k-mers, referred to as putative cis-regulatory elements (pCREs) are likely regulatory sequences of cold response in switchgrass. There are in total 655 pCREs where 54 are important in all cold treatment time points. Consistent with this, eight of 35 known cold-responsive CREs were similar to top-ranked pCREs in the models and only these eight were important for predicting temporal cold response. More importantly, most of the top-ranked pCREs were novel sequences in cold regulation. Our findings suggest additional sequence elements important for cold-responsive regulation previously not known that warrant further studies.

Transcriptome Analysis and SNP Identification Reveal That Heterologous Overexpression of Two Uncharacterized Genes Enhances the Tolerance of Magnaporthe oryzae to Manganese Toxicity.

ABSTRACTManganese is a crucial trace element that constitutes the cofactors of many enzymes. However, excessive Mn2+ can be toxic for both prokaryotes and eukaryotes. The mechanism of fungal genetics and metabolism in response to Mn2+ stress remains understudied, warranting further studies. Magnaporthe oryzae is well-established as the most destructive pathogen of rice. A field strain, YN2046, more sensitive to Mn2+ toxicity than other strains, was obtained from a previous study. Herein, we explored the genetic mechanisms of Mn2+ sensitivity in YN2046 through comparative transcriptomic analyses. We found that many genes previously reported to participate in Mn2+ stress were not regulated in YN2046. These non-responsive genes might cause Mn2+ sensitivity in YN2046. Weight gene correlation network analysis (WGCNA) was performed to characterize the expression profile in YN2046. Some overexpressed genes were only found in the Mn2+ tolerant isolate YN125. Among these, many single nucleotide polymorphism (SNP) were identified between YN125 and YN2046, which might disrupt the expression levels of Mn responsive genes. We cloned two uncharacterized genes, MGG_13347 and MGG_16609, from YN125 and transformed them to YN2046 with a strong promoter. Our results showed that the heterologous overexpression of two genes in YN2046 restored its sensitivity. Transcriptomic and biochemical analyses were performed to understand Mn tolerance mechanisms mediated by the two heterologous overexpressed genes. Our results showed that heterologous overexpression of these two genes activated downstream gene expression and metabolite production to restore M. oryzae sensitivity to Mn, implying that SNPs in responsive genes account for different phenotypes of the two strains under Mn stress.IMPORTANCE Heavy metals are used for fungicides as they target phytopathogen in multiple ways. Magnaporthe oryzae is the most destructive rice pathogen and is threatening global rice production. In the eukaryotes, the regulation mechanisms of Mn homeostasis often focus on the posttranslation, there were a few results about regulation at transcript level. The comparative transcriptome analysis showed that fewer genes were regulated in the Mn-sensitive strain. WGCNA and SNP analyses found that mutations in promoter and coding sequence regions might disrupt the expression of genes involved in Mn detoxification in the sensitive strain. We transferred two unannotated genes that were cloned from the Mn-tolerant strain into a sensitive strain with strong promoters, and the transformants exhibited an enhanced tolerance to Mn2+ toxicity. Transcriptome and biochemistry results indicated that heterologous overexpression of the two genes enhanced the tolerance to Mn toxicity by reactivation of downstream genes in M. oryzae.

Mysteries of gene regulation: Promoters are not the sole triggers of gene expression

Cis-regulatory elements of promoters are essential for gene regulation by transcription factors (TFs). However, the regulatory roles of nonpromoter regions, TFs, and epigenetic marks remain poorly understood in plants. In this study, we characterized the cis-regulatory regions of 53 TFs and 19 histone marks in 328 chromatin immunoprecipitation (ChIP-seq) datasets from Arabidopsis. The genome-wide maps indicated that both promoters and regions around the transcription termination sites of protein-coding genes recruit the most TFs. The maps also revealed a diverse of histone combinations. The analysis suggested that exons play critical roles in the regulation of non-coding genes. Additionally, comparative analysis between heat-stress-responsive and nonresponsive genes indicated that the genes with distinct functions also exhibited substantial differences in cis-regulatory regions, histone regulation, and topologically associating domain (TAD) boundary organization. By integrating multiple high-throughput sequencing datasets, this study generated regulatory models for protein-coding genes, non-coding genes, and TAD boundaries to explain the complexity of transcriptional regulation.

Volatiles from the fungal phytopathogen Penicillium aurantiogriseum modulate root metabolism and architecture through proteome resetting.

Volatile compounds (VCs) emitted by the fungal phytopathogen Penicillium aurantiogriseum promote root growth and developmental changes in Arabidopsis. Here we characterised the metabolic and molecular responses of roots to fungal volatiles. Proteomic analyses revealed that these compounds reduce the levels of aquaporins, the iron carrier IRT1 and apoplastic peroxidases. Fungal VCs also increased the levels of enzymes involved in the production of mevalonate (MVA)-derived isoprenoids, nitrogen assimilation and conversion of methionine to ethylene and cyanide. Consistently, fungal VC-treated roots accumulated high levels of hydrogen peroxide (H2 O2 ), MVA-derived cytokinins, ethylene, cyanide and long-distance nitrogen transport amino acids. qRT-PCR analyses showed that many proteins differentially expressed by fungal VCs are encoded by VC non-responsive genes. Expression patterns of hormone reporters and developmental characterisation of mutants provided evidence for the involvement of cyanide scavenging and enhanced auxin, ethylene, cytokinin and H2 O2 signalling in the root architecture changes promoted by fungal VCs. Our findings show that VCs from P. aurantiogriseum modify root metabolism and architecture, and improve nutrient and water use efficiencies through transcriptionally and non-transcriptionally regulated proteome resetting mechanisms. Some of these mechanisms are subject to long-distance regulation by photosynthesis and differ from those triggered by VCs emitted by beneficial microorganisms.

ELONGATED HYPOCOTYL 5 mediates blue light signalling to the Arabidopsis circadian clock.

Circadian clocks are gene networks producing 24-h oscillations at the level of clock gene expression that are synchronized to environmental cycles via light signals. The ELONGATED HYPOCOTYL 5 (HY5) transcription factor is a signalling hub acting downstream of several photoreceptors and is a key mediator of photomorphogenesis. Here we describe a mechanism by which light quality could modulate the pace of the circadian clock through governing abundance of HY5. We show that hy5 mutants display remarkably shorter period rhythms in blue but not in red light or darkness, and blue light is more efficient than red to induce accumulation of HY5 at transcriptional and post-transcriptional levels. We demonstrate that the pattern and level of HY5 accumulation modulates its binding to specific promoter elements of the majority of clock genes, but only a few of these show altered transcription in the hy5 mutant. Mathematical modelling suggests that the direct effect of HY5 on the apparently non-responsive clock genes could be masked by feedback from the clock gene network. We conclude that the information on the ratio of blue and red components of the white light spectrum is decoded and relayed to the circadian oscillator, at least partially, by HY5.

Methyl Jasmonate Regulates Podophyllotoxin Accumulation in Podophyllum hexandrum by Altering the ROS-Responsive Podophyllotoxin Pathway Gene Expression Additionally through the Down Regulation of Few Interfering miRNAs.

Podophylloxin (ptox), primarily obtained from Podophyllum hexandrum, is the precursor for semi-synthetic anticancer drugs viz. etoposide, etopophos, and teniposide. Previous studies established that methyl jasmonate (MeJA) treated cell culture of P. hexandrum accumulate ptox significantly. However, the molecular mechanism of MeJA induced ptox accumulation is yet to be explored. Here, we demonstrate that MeJA induces reactive oxygen species (ROS) production, which stimulates ptox accumulation significantly and up regulates three ROS-responsive ptox biosynthetic genes, namely, PhCAD3, PhCAD4 (cinnamyl alcohol dehydrogenase), and NAC3 by increasing their mRNA stability. Classic uncoupler of oxidative phosphorylation, carbonylcyanide m-chlorophenylhydrazone, as well as H2O2 treatment induced the ROS generation and consequently, enhanced the ptox production. However, when the ROS was inhibited with NADPH oxidase inhibitor diphenylene iodonium and Superoxide dismutase inhibitor diethyldithio-carbamic acid, the ROS inhibiting agent, the ptox production was decreased significantly. We also noted that, MeJA up regulated other ptox biosynthetic pathway genes which are not affected by the MeJA induced ROS. Further, these ROS non-responsive genes were controlled by MeJA through the down regulation of five secondary metabolites biosynthesis specific miRNAs viz. miR172i, miR035, miR1438, miR2275, and miR8291. Finally, this study suggested two possible mechanisms through which MeJA modulates the ptox biosynthesis: primarily by increasing the mRNA stability of ROS-responsive genes and secondly, by the up regulation of ROS non-responsive genes through the down regulation of some ROS non-responsive miRNAs.

Comprehensive analysis of promoter-proximal RNA polymerase II pausing across mammalian cell types.

BackgroundFor many genes, RNA polymerase II stably pauses before transitioning to productive elongation. Although polymerase II pausing has been shown to be a mechanism for regulating transcriptional activation, the extent to which it is involved in control of mammalian gene expression and its relationship to chromatin structure remain poorly understood.ResultsHere, we analyze 85 RNA polymerase II chromatin immunoprecipitation (ChIP)-sequencing experiments from 35 different murine and human samples, as well as related genome-wide datasets, to gain new insights into the relationship between polymerase II pausing and gene regulation. Across cell and tissue types, paused genes (pausing index > 2) comprise approximately 60 % of expressed genes and are repeatedly associated with specific biological functions. Paused genes also have lower cell-to-cell expression variability. Increased pausing has a non-linear effect on gene expression levels, with moderately paused genes being expressed more highly than other paused genes. The highest gene expression levels are often achieved through a novel pause-release mechanism driven by high polymerase II initiation. In three datasets examining the impact of extracellular signals, genes responsive to stimulus have slightly lower pausing index on average than non-responsive genes, and rapid gene activation is linked to conditional pause-release. Both chromatin structure and local sequence composition near the transcription start site influence pausing, with divergent features between mammals and Drosophila. Most notably, in mammals pausing is positively correlated with histone H2A.Z occupancy at promoters.ConclusionsOur results provide new insights into the contribution of RNA polymerase II pausing in mammalian gene regulation and chromatin structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0984-2) contains supplementary material, which is available to authorized users.

Rrd1p, an RNA Polymerase II‐specific Prolyl Isomerase and Activator of Phosphoprotein Phosphatase, Promotes Transcription Independently of Rapamycin Response

Rrd1p (resistance to rapamycin deletion 1) has been previously implicated in controlling transcription of rapamycin-regulated genes in response to rapamycin treatment. Intriguingly, we show here that Rrd1p associates with the coding sequence of a galactose-inducible and rapamycin non-responsive GAL1 gene, and promotes the association of RNA polymerase II with GAL1 in the absence of rapamycin treatment following transcriptional induction. Consistently, nucleosomal disassembly at GAL1 is impaired in the absence of Rrd1p, and GAL1 transcription is reduced in the Δrrd1 strain. Likewise, Rrd1p associates with the coding sequences of other rapamycin non-responsive and inducible GAL genes to promote their transcription in the absence of rapamycin treatment. Similarly, inducible, but rapamycin-responsive, non-GAL genes such as CTT1, STL1 and CUP1 are also regulated by Rrd1p. However, transcription of these inducible GAL and non-GAL genes is not altered in the absence of Rrd1p when the steady-state is reached after long transcriptional induction. Consistently, transcription of the constitutively active genes is not changed in the Δrrd1 strain. Taken together, our results demonstrate a new function of Rrd1p in stimulation of initial rounds of transcription, but not steady-state/constitutive transcription, of both rapamycin-responsive and non-responsive genes independently of rapamycin treatment.

Identification and expression analysis of genes responsive to drought stress in peanut

Although peanut (Arachis hypogaea L.) is a relatively drought-tolerant crop, its productivity and quality is adversely affected by drought stress. The identification of novel genes, the analysis of gene expression patterns in response to dehydration stress, and the evaluation of their potential functions in stress adaptation will provide the basis for effective engineering strategies to improve crop drought tolerance. However, little is known about stress tolerance of peanut. To investigate genes that are involved in the response to drought stress in peanut, a survey of genes responsive to drought stress was done by cDNA macroarray analysis, and about 277 unigenes were selected, including genes related among others to stress response and transcription factors. Totally, it was found that about 21 clones could respond to dehydration. The expression patterns of these 21 candidate drought-responsive genes and another 30 selected non-responsive genes were further analyzed by real-time PCR, and then we found another responsive gene P3617. Among these genes, 18 genes were up-regulated, and 4 genes were down-regulated by drought. The functional classification indicated that these genes belonged to gene families encoding stress-responsive proteins, transcription factors, auxin-responsive protein, Na+/H+ transporter SOS1, and chloroplast photosynthetic oxygen-evolving protein subunit of peanut. Collectively, the data presented in this study will facilitate the understanding of molecular mechanism of the peanut response to drought stress and also provide the potential strategies for effective genetic engineering to improve peanut stress tolerance.

Rrd1p, an RNA polymerase II-specific prolyl isomerase and activator of phosphoprotein phosphatase, promotes transcription independently of rapamycin response.

Rrd1p (resistance to rapamycin deletion 1) has been previously implicated in controlling transcription of rapamycin-regulated genes in response to rapamycin treatment. Intriguingly, we show here that Rrd1p associates with the coding sequence of a galactose-inducible and rapamycin non-responsive GAL1 gene, and promotes the association of RNA polymerase II with GAL1 in the absence of rapamycin treatment following transcriptional induction. Consistently, nucleosomal disassembly at GAL1 is impaired in the absence of Rrd1p, and GAL1 transcription is reduced in the Δrrd1 strain. Likewise, Rrd1p associates with the coding sequences of other rapamycin non-responsive and inducible GAL genes to promote their transcription in the absence of rapamycin treatment. Similarly, inducible, but rapamycin-responsive, non-GAL genes such as CTT1, STL1 and CUP1 are also regulated by Rrd1p. However, transcription of these inducible GAL and non-GAL genes is not altered in the absence of Rrd1p when the steady-state is reached after long transcriptional induction. Consistently, transcription of the constitutively active genes is not changed in the Δrrd1 strain. Taken together, our results demonstrate a new function of Rrd1p in stimulation of initial rounds of transcription, but not steady-state/constitutive transcription, of both rapamycin-responsive and non-responsive genes independently of rapamycin treatment.

Intragenic Controls Utilizing Radiation-Induced Alternative Transcript Regions Improves Gene Expression Biodosimetry

Ionizing-radiation exposure can be life threatening if given to the whole body. In addition, whole body radiation exposure can affect large numbers of people such as after a nuclear reactor accident, a nuclear explosion or a radiological terrorist attack. In these cases, an accurate biodosimeter is essential for triage management. One of the problems for biodosimetry in general is the interindividual variation before and after exposure, which can make it challenging to assign an accurate dose. To begin to address this challenge, lymphocyte cell lines were exposed to 0, 1, 2 and 5 Gy ionizing radiation from a ¹³⁷Cs source at a dose rate of 0.6 Gy/min. Alternative transcripts with regions showing large differential responses to ionizing radiation were determined from exon array data. Gene expression analysis was then performed on isolated mRNA using qRT-PCR with normalization to intergenic (PGK1, GAPDH) and novel intragenic regions for candidate radiation-responsive genes, PPM1D and MDM2. Our studies show that the use of a cis-associated expression reference improved the potential dose prediction approximately 2.3-8.3 fold and provided an advantage for dose prediction compared to distantly or trans-located control ionizing radiation nonresponsive genes. This approach also provides an alternative gene expression normalization method to potentially reduce interindividual variations when untreated basal gene expression levels are unavailable. Using associated noninduced regions of ionizing radiation-induced genes provides a way to estimate basal gene expression in the irradiated sample. This strategy can be utilized as a biodosimeter on its own or to enhance other gene expression candidates for biodosimetry. This normalization strategy may also be generally applicable for other quantitative PCR strategies where normalization is required for a particular response.

The differentially regulated genes TvQR1 and TvPirin of the parasitic plant Triphysaria exhibit distinctive natural allelic diversity

BackgroundPlant parasitism represents an extraordinary interaction among flowering plants: parasitic plants use a specialized organ, the haustorium, to invade the host vascular system to deprive host plants of water and nutrients. Various compounds present in exudates of host plants trigger haustorium development. The two most effective haustorium inducing factors (HIFs) known for the parasitic plant Triphysaria versicolor (T. versicolor) are peonidin, an antioxidant flavonoid, and 2,6-dimethoxybenzoquinone (DMBQ), an oxidative stress agent. To date, two genes involved in haustorium initiation in T. versicolor have been identified: TvQR1, a quinone oxidoreductase that generates the active HIF from DMBQ, and TvPirin, a transcription co-factor that regulates several other DMBQ- responsive and –non-responsive genes. While the expression of these genes in response to DMBQ is well characterized, their expression in response to peonidin is not. In addition, the pattern of polymorphisms in these genes is unknown, even though nucleotide changes in TvQR1 and TvPirin may have contributed to the ability of T. versicolor to develop haustoria. To gain insights into these aspects, we investigated their transcriptional responses to HIFs and non-HIF and their natural nucleotide diversity.ResultsHere we show that TvQR1 and TvPirin are transcriptionally upregulated by both DMBQ and peonidin in T. versicolor roots. Yet, while TvQR1 also responded to juglone, a non-HIF quinone with toxicity comparable to that of DMBQ, TvPirin did not. We further demonstrate that TvPirin encodes a protein shorter than the one previously reported. In the T. versicolor natural population of Northern California, TvQR1 exhibited remarkably higher molecular diversity and more recombination events than TvPirin, with the highest non-synonymous substitution rate in the substrate recognition and catalytic domain of the TvQR1 protein.ConclusionOur results suggest that TvQR1 and TvPirin have most likely evolved highly distinct roles for haustorium formation. Unlike TvPirin, TvQR1 might have been under diversifying selection to maintain a diverse collection of polymorphisms, which might be related to the recognition of an assortment of HIF and non-HIF quinones as substrates for successful haustorial establishment in a wide range of host plants.

Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity

BackgroundIn addition to determining static states of gene expression (high vs. low), it is important to characterize their dynamic status. For example, genes with H3K27me3 chromatin marks are not only suppressed but also poised for activation. However, the responsiveness of genes to perturbations has never been studied systematically. To distinguish gene responses to specific factors from responsiveness in general, it is necessary to analyze gene expression profiles of cells responding to a large variety of disturbances, and such databases did not exist before.ResultsWe estimated the responsiveness of all genes in mouse ES cells using our recently published database on expression change after controlled induction of 53 transcription factors (TFs) and other genes. Responsive genes (N = 4746), which were readily upregulated or downregulated depending on the kind of perturbation, mostly have regulatory functions and a propensity to become tissue-specific upon differentiation. Tissue-specific expression was evaluated on the basis of published (GNF) and our new data for 15 organs and tissues. Non-responsive genes (N = 9562), which did not change their expression much following any perturbation, were enriched in housekeeping functions. We found that TF-responsiveness in ES cells is the best predictor known for tissue-specificity in gene expression. Among genes with CpG islands, high responsiveness is associated with H3K27me3 chromatin marks, and low responsiveness is associated with H3K36me3 chromatin, stronger tri-methylation of H3K4, binding of E2F1, and GABP binding motifs in promoters.ConclusionsWe thus propose the responsiveness of expression to perturbations as a new way to define the dynamic status of genes, which brings new insights into mechanisms of regulation of gene expression and tissue specificity.

Up-regulation of competence- but not stress-responsive proteins accompanies an altered metabolic phenotype in Streptococcus mutans biofilms

Mature biofilm and planktonic cells of Streptococcus mutans cultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0.050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess a cin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms of S. mutans in a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.

Genetic diversity of drought-responsive genes in populations of the desert forage Dactylis glomerata

Restriction fragment length polymorphisms were used to estimate genetic diversity in six populations of Dactlylis glomerata collected in two different geographical locations in Israel. The objective of this study was to test the hypothesis that drought-responsive genes would have a higher genetic diversity in populations adapted to arid environments. Drought-responsive and non-responsive genes were isolated as cDNAs from leaves of D. glomerata. These cDNAs were used as probes on genomic Southern blots. Populations of D. glomerata collected in the southern arid region of Israel had greater genetic diversity for drought-responsive genes (0.388, repressed; 0.340, induced) than populations of plants collected in the northern Mediterranean region of Israel (0.308, repressed; 0.314, induced). The populations collected in the Mediterranean region had greater genetic diversity for the genes that were non-responsive to drought (0.185 versus 0.086). The gene flow between populations was relatively high, 2.47 for induced, 1.96 for repressed and 3.5 for non-responsive genes. These results suggest that plant conservation analyses should also consider stress responsive genes in estimates of genetic diversity, to select populations for conservation.

Epstein-Barr virus SM protein interacts with mRNA in vivo and mediates a gene-specific increase in cytoplasmic mRNA.

SM is an Epstein-Barr virus (EBV) gene expressed during early lytic replication of EBV. SM encodes a nuclear phosphoprotein that functions as a posttranscriptional regulator of gene expression. SM has been implicated in several aspects of gene regulation, including nuclear mRNA stabilization, posttranscriptional processing, and nuclear mRNA export. Activation by SM is promoter independent but gene specific. The mechanism by which SM selectively activates some EBV target genes or heterologous reporter genes remains to be determined. SM binds RNA in vitro, suggesting that sequence- or structure-specific mRNA interactions might mediate SM specificity. We have further analyzed RNA binding by SM and demonstrated that proteolytic cleavage of SM and consequent exposure of an arginine-rich region are necessary to allow RNA binding in vitro. However, SM mutants with deletions of this arginine-rich region localized normally in the nucleus and were fully functional in gene activation. We therefore developed an assay to study in vivo interactions of SM with target mRNAs based on immunoprecipitation of SM from cell lysates followed by RNase protection analysis. Using this assay, we demonstrated that SM forms complexes with specific mRNAs in vivo. SM binds mRNAs from both SM-responsive as well as nonresponsive intronless genes and increases the nuclear accumulation of both types of mRNAs. In addition, SM preferentially associates with newly transcribed mRNAs. These data indicate that SM forms complexes with mRNAs in the nucleus and enhances their nuclear accumulation. However, SM does not enhance cytoplasmic accumulation of all transcripts that it binds to the same degree, suggesting that additional mRNA-specific characteristics, such as nuclear retention motifs or binding sites for cellular proteins, also determine responsiveness to SM.

Dysregulated expression of androgen-responsive and nonresponsive genes in the androgen-independent prostate cancer xenograft model, CWR22-R

Dysregulated expression of androgen-responsive and nonresponsive genes in the androgen-independent prostate cancer xenograft model, CWR22-R

Promoter elements of the PHR1 gene of Saccharomyces cerevisiae and their roles in the response to DNA damage.

The PHR1 gene of Saccharomyces cerevisiae encodes the apoenzyme for the DNA repair enzyme photolyase. PHR1 transcription is induced in response to 254 nm radiation and a variety of chemical damaging agents. We report here the identification of promoter elements required for PHR1 expression. Transcription is regulated primarily through three sequence elements clustered within a 120 bp region immediately upstream of the translational start site. A 20 bp interrupted palindrome comprises UASPHR1 and is responsible for 80-90% of basal and induced expression. UASPHR1 alone can activate transcription of a CYC1 minimal promoter but does not confer damage responsiveness. In the intact PHR1 promoter UAS function is dependent upon an upstream essential sequence (UES). URSPHR1 contains a binding site for the damage-responsive repressor Prp; consistent with this role, deletion or specific mutations of the URS increase basal level expression and decrease the induction ratio. Deletion of URSPHR1 also eliminates the requirement for UESPHR1 for promoter activation, indicating that the UES attenuates Prp-mediated repression. Sequences within UASPHR1 are similar to regulatory sequences found upstream of both damage responsive and nonresponsive genes involved in DNA repair and metabolism.

Characterization of paralogous and orthologous members of the superoxide dismutase gene family from genera of the halophilic archaebacteria.

Four species representing three genera of halophilic archaebacteria were examined for the presence of genomic sequences that encode proteins of the superoxide dismutase family. Three species, Halobacterium cutirubrum, Halobacterium sp. strain GRB, and Haloferax volcanii, contain duplicated (paralogous) genes of the sod family; a fourth species, Haloarcula marismortui, contains only a single gene. These seven genes were cloned and sequenced, and their transcripts were characterized by Northern (RNA) hybridization, S1 nuclease protection, and primer extension. The expression of one of the two genes in H. cutirubrum, Halobacterium sp. strain GRB, and Haloferax volcanii was shown to be elevated in the presence of paraquat, a generator of superoxide radicals. The other genes, including the single gene from Haloarcula marismortui, exhibited no elevated expression in the presence of paraquat. The 5' and 3' flanking regions of all the genes contain recognizable promoter and terminator elements that are appropriately positioned relative to the 5' and 3' transcript end sites. Between genera, the orthologous paraquat-responsive genes exhibit no sequence similarity in either their 5' or 3' flanking regions, whereas the orthologous nonresponsive genes exhibit limited sequence similarity but only in the 5' flanking region. Within the coding region, the two paralogous genes of Haloferax volcanii are virtually identical (99.5%) despite the absence of similarity in the flanking regions. In contrast, the paralogous genes of H. cutirubrum and Halobacterium sp. strain GRB are only about 87% identical. In the alignment of all seven sequences, there are nine codon positions where both the TCN and AGY serine codons are utilized; some or all of these may well be examples of convergent evolution.