Transcription factories : gene expression in unions?

Transcription is a fundamental step in gene expression, yet it remains poorly understood at a cellular level. Visualization of transcription sites and active genes has led to the suggestion that transcription occurs at discrete sites in the nucleus, termed transcription factories, where multiple active RNA polymerases are concentrated and anchored to a nuclear substructure. However, this concept is not universally accepted. This Review discusses the experimental evidence in support of the transcription factory model and the evidence that argues against such a spatially structured view of transcription. The transcription factory model has implications for the regulation of transcription initiation and elongation, for the organization of genes in the genome, for the co-regulation of genes and for genome instability.

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

Oestrogen-modulated gene expression in the human endometrium

To identify key regulatory mechanisms in the growth and development of the human endometrium, microarray analysis was performed on uncultured human endometrium collected during menstruation (M) and the late-proliferative (LATE-P)-phase of the menstrual cycle, as well as after 24 h incubation in the presence of oestradiol (17beta-E2). We demonstrate the expression of novel gene transcripts in the human endometrium. i.e. mucin-9, novel oestrogen-responsive gene transcripts, i.e. gelsolin and flotillin-1, and genes known to be expressed in human endometrium but not yet shown to be oestrogen responsive, i.e. connexin-37 and TFF1/pS2. Genes reported to be expressed during the implantation window and implicated in progesterone action, i.e. secretoglobin family 2A, member 2 (mammaglobin) and homeobox-containing proteins, were up-regulated in uncultured LATE-P-phase endometrium compared to M-phase endometrium. Some gene transcripts are regulated directly by 17beta-E2 alone, others are influenced by the in vivo environment as well. These observations emphasise that the regulation of endometrium maturation by oestrogen entails more then just stimulation of cell proliferation.

Estrogen and the endometrium: lessons learned from gene expression profiling in rodents and human

To date, research into the biological processes and molecular mechanisms associated with endometrial receptivity and embryo implantation has been a focus of attention, whereas the complex events that occur in the human endometrium during the menstrual and proliferative phase under the influence of estrogen have received little attention. The objective of this review is to provide an update of our current understanding of the actions of estrogen on both human and rodent endometrium, with special emphasis on the regulation of uterine growth and cell proliferation, and the value of global gene expression analysis, in increasing understanding of these processes.

Edge-weighting of gene expression graphs

In recent years, considerable research efforts have been directed to micro-array technologies and their role in providing simultaneous information on expression profiles for thousands of genes. These data, when subjected to clustering and classification procedures, can assist in identifying patterns and providing insight on biological processes. To understand the properties of complex gene expression datasets, graphical representations can be used. Intuitively, the data can be represented in terms of a bipartite graph, with weighted edges corresponding to gene-sample node couples in the dataset. Biologically meaningful subgraphs can be sought, but performance can be influenced both by the search algorithm, and, by the graph-weighting scheme and both merit rigorous investigation. In this paper, we focus on edge-weighting schemes for bipartite graphical representation of gene expression. Two novel methods are presented: the first is based on empirical evidence; the second on a geometric distribution. The schemes are compared for several real datasets, assessing efficiency of performance based on four essential properties: robustness to noise and missing values, discrimination, parameter influence on scheme efficiency and reusability. Recommendations and limitations are briefly discussed. Keywords: Edge-weighting; weighted graphs; gene expression; bi-clustering

Gene expression profiling reveals a downregulation in immune-associated genes in patients with AS

Objective: To identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) compared with healthy individuals. Methods: RNA was extracted from PBMCs collected from 18 patients with active disease and 18 gender-matched and age-matched controls. Expression profiles of these cells were determined using microarray. Candidate genes with differential expressions were confirmed in the same samples using quantitative reverse transcription-PCR (qRT-PCR). These genes were then validated in a different sample cohort of 35 patients with AS and 18 controls by qRT-PCR. Results: Microarray analysis identified 452 genes detected with 485 probes which were differentially expressed between patients with AS and controls. Underexpression of NR4A2, tumour necrosis factor AIP3 (TNFAIP3) and CD69 was confirmed. These genes were further validated in a different sample group in which the patients with AS had a wider range of disease activity. Predictive algorithms were also developed from the expression data using receiver-operating characteristic curves, which demonstrated that the three candidate genes have ∼80% power to predict AS according to their expression levels. Conclusions: The findings show differences in global gene expression patterns between patients with AS and controls, suggesting an immunosuppressive phenotype in the patients. Furthermore, downregulated expression of three immune-related genes was confirmed. These candidate genes were also shown to be strong predictive markers for AS.

Genetic association and gene expression studies suggest that genetic variants in the SYNE1 and TNF genes are related to menstrual migraine

BACKGROUND: Menstrual migraine (MM) encompasses pure menstrual migraine (PMM) and menstrually-related migraine (MRM). This study was aimed at investigating genetic variants that are potentially related to MM, specifically undertaking genotyping and mRNA expression analysis of the ESR1, PGR, SYNE1 and TNF genes in MM cases and non-migraine controls. METHODS: A total of 37 variants distributed across 14 genes were genotyped in 437 DNA samples (282 cases and 155 controls). In addition levels of gene expression were determined in 74 cDNA samples (41 cases and 33 controls). Association and correlation analysis were performed using Plink and RStudio. RESULTS: SNPs rs3093664 and rs9371601 in TNF and SYNE1 genes respectively, were significantly associated with migraine in the MM population (p = 0.008; p = 0.009 respectively). Analysis of qPCR results found no significant difference in levels of gene expression between cases and controls. However, we found a significant correlation between the expression of ESR1 and SYNE1, ESR1 and PGR and TNF and SYNE1 in samples taken during the follicular phase of the menstrual cycle. CONCLUSIONS: Our results show that SNPs rs9371601 and rs3093664 in the SYNE1 and TNF genes respectively, are associated with MM. The present study also provides strong evidence to support the correlation of ESR1, PGR, SYNE1 and TNF gene expression in MM.

Analysis of differential gene expression based on Bayesian estimation of variance

Gene expression is arguably the most important indicator of biological function. Thus identifying differentially expressed genes is one of the main aims of high throughout studies that use microarray and RNAseq platforms to study deregulated cellular pathways. There are many tools for analysing differentia gene expression from transciptomic datasets. The major challenge of this topic is to estimate gene expression variance due to the high amount of ‘background noise’ that is generated from biological equipment and the lack of biological replicates. Bayesian inference has been widely used in the bioinformatics field. In this work, we reveal that the prior knowledge employed in the Bayesian framework also helps to improve the accuracy of differential gene expression analysis when using a small number of replicates. We have developed a differential analysis tool that uses Bayesian estimation of the variance of gene expression for use with small numbers of biological replicates. Our method is more consistent when compared to the widely used cyber-t tool that successfully introduced the Bayesian framework to differential analysis. We also provide a user-friendly web based Graphic User Interface for biologists to use with microarray and RNAseq data. Bayesian inference can compensate for the instability of variance caused when using a small number of biological replicates by using pseudo replicates as prior knowledge. We also show that our new strategy to select pseudo replicates will improve the performance of the analysis. - See more at: http://www.eurekaselect.com/node/138761/article#sthash.VeK9xl5k.dpuf

The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

Role of TATATAA element in the regulation of tRNA(1)(Gly) gene expression in Bombyx mori is position dependent

Transcription of tRNA genes by RNA polymerase III is controlled by the internal conserved sequences within the coding region and the immediate upstream flanking sequences. A highly transcribed copy of glycyl tRNA gene tRNA1(Gly)-1 from Bombyx mori is down regulated by sequences located much farther upstream in the region -150 to -300 nucleotides (nt), with respect to the +1 nt of tRNA. The negative regulatory effect has been narrowed down to a sequence motif 'TATATAA', a perfect consensus recognised by the TATA binding protein, TBP. This sequence element, when brought closer to the transcription start point, on the other hand, exerts a positive effect by promoting transcription of the gene devoid of other cis regulatory elements. The identity of the nuclear protein interacting with this 'TATATAA' element to TBP has been established by antibody and mutagenesis studies. The 'TATATAA' element thus influences the transcription of tRNA genes positively or negatively in a position-dependent manner either by recruitment or sequestration of TBP from the transcription machinery.

Genome-Wide Gene Expression Analysis Reveals a Dynamic Interplay between Luteotropic and Luteolytic Factors in the Regulation of Corpus Luteum Function in the Bonnet Monkey (Macaca radiata)

Although LH is essential for survival and function of the corpus luteum (CL) in higher primates, luteolysis occurs during nonfertile cycles without a discernible decrease in circulating LH levels. Using genome-wide expression analysis, several experiments were performed to examine the processes of luteolysis and rescue of luteal function in monkeys. Induced luteolysis with GnRH receptor antagonist (Cetrorelix) resulted in differential regulation of 3949 genes, whereas replacement with exogenous LH (Cetrorelix plus LH) led to regulation of 4434 genes (1563 down-regulation and 2871 up-regulation). A model system for prostaglandin (PG) F-2 alpha-induced luteolysis in the monkey was standardized and demonstrated that PGF(2 alpha) regulated expression of 2290 genes in the CL. Analysis of the LH-regulated luteal transcriptome revealed that 120 genes were regulated in an antagonistic fashion by PGF(2 alpha). Based on the microarray data, 25 genes were selected for validation by real-time RT-PCR analysis, and expression of these genes was also examined in the CL throughout the luteal phase and from monkeys treated with human chorionic gonadotropin (hCG) to mimic early pregnancy. The results indicated changes in expression of genes favorable to PGF(2 alpha) action during the late to very late luteal phase, and expressions of many of these genes were regulated in an opposite manner by exogenous hCG treatment. Collectively, the findings suggest that curtailment of expression of downstream LH-target genes possibly through PGF(2 alpha) action on the CL is among the mechanisms underlying cross talk between the luteotropic and luteolytic signaling pathways that result in the cessation of luteal function, but hCG is likely to abrogate the PGF(2 alpha)-responsive gene expression changes resulting in luteal rescue crucial for the maintenance of early pregnancy. (Endocrinology 150: 1473-1484, 2009)

Regulation of Gonadotropin-Releasing Hormone (GnRH)-Receptor Gene Expression in Tilapia: Effect of GnRH and Dopamine

The present work was designed to study certain aspects of the endocrine regulation of gonadotropin-releasing hormone receptor (GnRH-R) in the pituitary of the teleost fish tilapia. A GnRH-R was cloned from the pituitary of hybrid tilapia (taGnRH-R) and was identified as a typical seven-transmembrane receptor. Northern blot analysis revealed a single GnRH-R transcript in the pituitary of approximately 2.3 kilobases. The taGnRH-R mRNA levels were significantly higher in females than in males. Injection of the salmon GnRH analog (sGnRHa; 5–50 μg/kg) increased the steady-state levels of taGnRH-R mRNA, with the highest response recorded at 25 μg/kg and at 36 h. At the higher dose of sGnRHa (50 μg/kg), taGnRH-R transcript appeared to be down-regulated. Exposure of tilapia pituitary cells in culture to graded doses (0.1–100 nM) of seabream (sbGnRH = GnRH I), chicken II (cGnRH II), or salmon GnRH (sGnRH = GnRH III) resulted in a significant increase in taGnRH-R mRNA levels. The highest levels of both LH release and taGnRH-R mRNA levels were recorded after exposure to cGnRH II and the lowest after exposure to sbGnRH. The dopamine-agonist quinpirole suppressed LH release and mRNA levels of taGnRH-R, indicating an inhibitory effect on GnRH-R synthesis. Collectively, these data provide evidence that GnRH in tilapia can up- regulate, whereas dopamine down-regulates, taGnRH-R mRNA levels.

Standardization and validation of an induced ovulation model system in buffalo cows: Characterization of gene expression changes in the periovulatory follicle

In bovines characterization of biochemical and molecular determinants of the dominant follicle before and during different time intervals after gonadotrophin surge requires precise identification of the dominant follicle from a follicular wave. The objectives of the present study were to standardize an experimental model in buffalo cows for accurately identifying the dominant follicle of the first wave of follicular growth and characterize changes in follicular fluid hormone concentrations as well as expression patterns of various genes associated with the process of ovulation. From the day of estrus (day 0), animals were subjected to blood sampling and ultrasonography for monitoring circulating progesterone levels and follicular growth. On day 7 of the cycle, animals were administered a PGF2α analogue (Tiaprost Trometamol, 750 μg i.m.) followed by an injection of hCG (2000 IU i.m.) 36 h later. Circulating progesterone levels progressively increased from day 1 of the cycle to 2.26 ± 0.17 ng/ml on day 7 of the cycle, but declined significantly after PGF2α injection. A progressive increase in the size of the dominant follicle was observed by ultrasonography. The follicular fluid estradiol and progesterone concentrations in the dominant follicle were 600 ± 16.7 and 38 ± 7.6 ng/ml, respectively, before hCG injection and the concentration of estradiol decreased to 125.8 ± 25.26 ng/ml, but concentration of progesterone increased to 195 ± 24.6 ng/ml, 24 h post-hCG injection. Inh-α and Cyp19A1 expressions in granulosa cells were maximal in the dominant follicle and declined in response to hCG treatment. Progesterone receptor, oxytocin and cycloxygenase-2 expressions in granulosa cells, regarded as markers of ovulation, were maximal at 24 h post-hCG. The expressions of genes belonging to the super family of proteases were also examined; Cathepsin L expression decreased, while ADAMTS 3 and 5 expressions increased 24 h post-hCG treatment. The results of the current study indicate that sequential treatments of PGF2α and hCG during early estrous cycle in the buffalo cow leads to follicular growth that culminates in ovulation. The model system reported in the present study would be valuable for examining temporo-spatial changes in the periovulatory follicle immediately before and after the onset of gonadotrophin surge.

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background: Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton. Results: We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. A total of 21 distinct moult-cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Of these, 13 contained copies of the cuticle_1 domain previously isolated from calcified regions of the crustacean exoskeleton, four transcripts contained a chitin_bind_4 domain (RR consensus sequence) associated with both the calcified and un-calcified cuticle of crustaceans, and four transcripts contained an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally, cryptocyanin, a hemolymph protein involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. Conclusion: The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening. This study provides a molecular entry path into the investigation of the gene networks associated with cuticle formation.

Gene expression in the skin of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus

The cattle tick Rhipicephalus microplus (formerly Boophilus microplus) is responsible for severe production losses to the cattle industry worldwide. It has long been known that different breeds of cattle can resist tick infestation to varying degrees; however, the mechanisms by which resistant cattle prevent heavy infestation are largely unknown. The aim of this study was to determine whether gene expression varied significantly between skin sampling sites (neck, chest and tail region), and whether changes in gene expression could be detected in samples taken at tick attachment sites (tick attached to skin sample) compared with samples taken from non-attachment sites (no tick attachment). We present here the results of an experiment examining the expression of a panel of forty-four genes in skin sections taken from Bos indicus (Brahman) cattle of known high resistance, and Bos taurus (Holstein-Friesian) cattle of known low resistance to the cattle tick. The forty-four genes chosen for this study included genes known to be involved in several immune processes, some structural genes, and some genes previously suggested to be of importance in tick resistance by other researchers. The expression of fifteen gene transcripts increased significantly in Holstein-Friesian skin samples at tick attachment sites. The higher expression of many genes involved in innate inflammatory processes in the Holstein-Friesian animals at tick attachment sites suggests this breed is exhibiting a non-directed pathological response to infestation. Of the forty-four genes analysed, no transcripts were detected in higher abundance at tick attachment sites in the Brahman cattle compared with similar samples from the Holstein-Friesian group, nor difference between attachment site and non-attachment site samples within the Brahman group. The results presented here suggest that the means by which these two cattle breeds respond to tick infestation differ and warrant further investigation.