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Coronavirus disease 2019 pandemic is the most damaging pandemic in recent human history. Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and variant strains is paramount for recovery from this pandemic. Conventional SARS-CoV-2 tests interrogate only limited regions of the whole SARS-CoV-2 genome, which are subjected to low specificity and miss the opportunity of detecting variant strains. In this work, we developed the first SARS-CoV-2 tiling array that captures the entire SARS-CoV-2 genome at single nucleotide resolution and offers the opportunity to detect point mutations. A thorough bioinformatics protocol of two base calling methods has been developed to accompany this array. To demonstrate the effectiveness of the tiling array, we genotyped all genomic positions of eight SARS-CoV-2 samples. Using high-throughput sequencing as the benchmark, we show that the tiling array had a genome-wide accuracy of at least 99.5%. From the tiling array analysis results, we identified the D614G mutation in the spike protein in four of the eight samples, suggesting the widespread distribution of this variant at the early stage of the outbreak in the United States. Two additional nonsynonymous mutations were identified in one sample in the nucleocapsid protein (P13L and S197L), which may complicate future vaccine development. With around $5 per array, supreme accuracy, and an ultrafast bioinformatics protocol, the SARS-CoV-2 tiling array makes an invaluable toolkit for combating current and future pandemics. Our SARS-CoV-2 tilting array is currently utilized by Molecular Vision, a CLIA-certified lab for SARS-CoV-2 diagnosis. [ABSTRACT FROM AUTHOR]

Fresh foods are vulnerable to foodborne pathogens which cause foodborne illness and endanger people's life and safety. The rapid detection of foodborne pathogens is crucial for food safety surveillance. An in situ -synthesized gene chip for the detection of foodborne pathogens on fresh-cut fruits and vegetables was developed. The target genes were identified and screened by comparing the specific sequences of Salmonella Typhimurium, Vibrio parahemolyticus , Staphylococcus aureus , Listeria monocytogenes , and Escherichia coli O157:H7 from the National Center for Biotechnology Information database. Tiling array probes were designed to target selected genes in an optimized hybridization system. A total of 141 specific probes were selected from 3,227 hybridization probes, comprising 26 L. monocytogenes , 24 S. aureus , 25 E. coli O157:H7, 20 Salmonella Typhimurium, and 46 V. parahemolyticus probes that are unique to this study. The optimized assay had strong amplification signals and high accuracy. The detection limit for the five target pathogens on fresh-cut cantaloupe and lettuce was approximately 3 log cfu/g without culturing and with a detection time of 24 h. The detection technology established in this study can rapidly detect and monitor the foodborne pathogens on fresh-cut fruits and vegetables throughout the logistical distribution chain, i.e., processing, cleaning, fresh-cutting, packaging, storage, transport, and sale, and represents a valuable technology that support the safety of fresh agricultural products. [ABSTRACT FROM AUTHOR]

Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of approximately 64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes-events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

Light plays a key role in the regulation of many physiological processes required for plant and chloroplast development. Plant cryptochromes (crys) play an important role in monitoring, capturing, and transmitting the light stimuli. In this study, we analyzed the effects of CRY2 overexpression on transcription of tomato chloroplast genome by a tiling array, containing about 90 000 overlapping probes (5‐nucleotide resolution). We profiled transcription in leaves of wild‐type and CRY2‐overexpressing plants grown in a diurnal cycle, to generate a comprehensive map of chloroplast transcription and to monitor potential specific modulations of the chloroplast transcriptome induced by the overexpression of CRY2. Our results demonstrate that CRY2 is a master gene of transcriptional regulation in the tomato chloroplast. In fact, it modulates the day/night mRNA abundance of about 58% of the 114 ORFs. The effect of CRY2 includes a differential extension of some transcripts at their 5′‐end, according to the period of the day. We observed that the influence of CRY2 on chloroplast transcription is not limited to coding RNA; a great number of putative noncoding micro RNA also showed differential accumulation pattern. To our knowledge, this is the first study that highlights how a photoreceptor affects the day/night transcription of the chloroplast genome.

Background: There are several techniques to analyse copy number variation in both research and clinical settings, such as whole genome amplification (sWGA), SNP arrays and one of the most commonly used techniques, array based comparative genomic hybridization (aCGH). In the latter, copy number comparison is obtained between differentially labelled target and reference DNAs by measuring ratio of fluorescence intensity of probes indicating loss or gain in the chromosomal region. Methods: Here we carry out a comparative analysis between two Plasmodium falciparum parasite isolates (Pf-isolate-2 and Pf-isolate-1) causing malaria using array CGH. The array contains approximately 418,577, 60mer custom-designed probes with an average probe spacing of 56 bp. The significant major variations (amplifications and deletions) copy number variations (CNV) in Pf-isolate-2 (Pf-2) in comparison with Pf-isolate-1 (Pf-1), are reported. Results: CNVs have been seen in all the chromosomes in Pf-2, most of the deletions have been seen mostly in sub-telomeric and telomeric regions of the chromosomes that comprises of variant surface antigen family genes. Apart from the subtelomeric regions other parts of the chromosomes have also shown CNVs. Novel variations , like continuous amplification of 28kb region (249817-278491) of chromosome-8, which covers for 3 genes two of which codes for conserved Plasmodium proteins with unknown function (MAL8P1.139, PF08_0122) and tRNA pseudouridine synthase, putative (PF08_0123). Amplifications in regions harboring genes like GTP cyclohydrolase I ( GCH-1, PFL1155W) and ribosomal protein, L24, putative (PFL1150C ) of chromosome 12 were seen. Conclusion: Other than known variations reported earlier, some novel variations have also been seen in the chromosomes of Pf-2. This is an experimental case study reporting major amplifications and deletions in Pf-isolate-2 in comparison with Pf-isolate-1 using a tiling array based comparative genomic hybridization approach.

Plants adapt to the prevailing photoperiod by adjusting growth and flowering to the availability of energy. To understand the molecular changes involved in adaptation to a long-day condition we comprehensively profiled leaf six at the end of the day and the end of the night at four developmental stages on Arabidopsis thaliana plants grown in a 16 h photoperiod, and compared the profiles to those from leaf 6 of plants grown in a 8 h photoperiod. When Arabidopsis is grown in a long-day photoperiod individual leaf growth is accelerated but whole plant leaf area is decreased because total number of rosette leaves is restricted by the rapid transition to flowering. Carbohydrate measurements in long- and short-day photoperiods revealed that a long photoperiod decreases the extent of diurnal turnover of carbon reserves at all leaf stages. At the transcript level we found that the long-day condition has significantly reduced diurnal transcript level changes than in short-day condition, and that some transcripts shift their diurnal expression pattern. Functional categorisation of the transcripts with significantly different levels in short and long day conditions revealed photoperiod-dependent differences in RNA processing and light and hormone signalling, increased abundance of transcripts for biotic stress response and flavonoid metabolism in long photoperiods, and for photosynthesis and sugar transport in short photoperiods. Furthermore, we found transcript level changes consistent with an early release of flowering repression in the long-day condition. Differences in protein levels between long and short photoperiods mainly reflect an adjustment to the faster growth in long photoperiods. In summary, the observed differences in the molecular profiles of leaf six grown in long- and short-day photoperiods reveal changes in the regulation of metabolism that allow plants to adjust their metabolism to the available light. The data also suggest that energy management is in the two photoperiods fundamentally different as a consequence of photoperiod-dependent energy constraints.

Light plays a key role in the regulation of many physiological processes required for plant and chloroplast development. Plant cryptochromes (crys) play an important role in monitoring, capturing, and transmitting the light stimuli. In this study, we analyzed the effects of CRY2 overexpression on transcription of tomato chloroplast genome by a tiling array, containing about 90 000 overlapping probes (5-nucleotide resolution). We profiled transcription in leaves of wild-type and CRY2-overexpressing plants grown in a diurnal cycle, to generate a comprehensive map of chloroplast transcription and to monitor potential specific modulations of the chloroplast transcriptome induced by the overexpression of CRY2. Our results demonstrate that CRY2 is a master gene of transcriptional regulation in the tomato chloroplast. In fact, it modulates the day/night mRNA abundance of about 58% of the 114 ORFs. The effect of CRY2 includes a differential extension of some transcripts at their 5′-end, according to the period of the day. We observed that the influence of CRY2 on chloroplast transcription is not limited to coding RNA; a great number of putative noncoding micro RNA also showed differential accumulation pattern. To our knowledge, this is the first study that highlights how a photoreceptor affects the day/night transcription of the chloroplast genome. [ABSTRACT FROM AUTHOR]

Emiliania huxleyi is the main calcite producer on Earth and is routinely infected by a virus (EhV); a double stranded DNA (dsDNA) virus belonging to the family Phycodnaviridae. E. huxleyi exhibits a haplodiploid life cycle; the calcified diploid stage is non-motile and forms extensive blooms. The haploid phase is a non-calcified biflagellated cell bearing organic scales. Haploid cells are thought to resist infection, through a process deemed the "Cheshire Cat"escape strategy; however, a recent study detected the presence of viral lipids in the same haploid strain. Here we report on the application of an E. huxleyi CCMP1516 EhV-86 combined tiling array (TA) that further confirms an EhV infection in the RCC1217 haploid strain, which grew without any signs of cell lysis. Reverse transcription polymerase chain reaction (RT-PCR) and PCR verified the presence of viral RNA in the haploid cells, yet indicated an absence of viral DNA, respectively. These infected cells are an alternative stage of the virus life cycle deemed the haplococcolithovirocell. In this instance, the host is both resistant to and infected by EhV, i.e., the viral transcriptome is present in haploid cells whilst there is no evidence of viral lysis. This superimposed state is reminiscent of Schrödinger's cat; of being simultaneously both dead and alive. [ABSTRACT FROM AUTHOR]

Abstract Despite the knowledge of complex prokaryotic‐transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well‐defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome‐wide characterization of transcript structures of ∼64% of all genes, including putative non‐coding RNAs in Halobacterium salinarum NRC‐1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment‐dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non‐functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements.

Emiliania huxleyi is the main calcite producer on Earth and is routinely infected by a virus (EhV); a double stranded DNA (dsDNA) virus belonging to the family Phycodnaviridae. E. huxleyi exhibits a haplodiploid life cycle; the calcified diploid stage is non-motile and forms extensive blooms. The haploid phase is a non-calcified biflagellated cell bearing organic scales. Haploid cells are thought to resist infection, through a process deemed the “Cheshire Cat” escape strategy; however, a recent study detected the presence of viral lipids in the same haploid strain. Here we report on the application of an E. huxleyi CCMP1516 EhV-86 combined tiling array (TA) that further confirms an EhV infection in the RCC1217 haploid strain, which grew without any signs of cell lysis. Reverse transcription polymerase chain reaction (RT-PCR) and PCR verified the presence of viral RNA in the haploid cells, yet indicated an absence of viral DNA, respectively. These infected cells are an alternative stage of the virus life cycle deemed the haplococcolithovirocell. In this instance, the host is both resistant to and infected by EhV, i.e., the viral transcriptome is present in haploid cells whilst there is no evidence of viral lysis. This superimposed state is reminiscent of Schrödinger’s cat; of being simultaneously both dead and alive.

Bacteria rapidly change their transcriptional patterns during infection in order to adapt to the host environment. To investigate host-bacteria interactions, various strategies including the use of animal infection models, in vitro assay systems and microscopic observations have been used. However, these studies primarily focused on a few specific genes and molecules in bacteria. High-density tiling arrays and massively parallel sequencing analyses are rapidly improving our understanding of the complex host-bacterial interactions through identification and characterization of bacterial transcriptomes. Information resulting from these high-throughput techniques will continue to provide novel information on the complexity, plasticity and regulation of bacterial transcriptomes as well as their adaptive responses relative to pathogenecity. Here we summarize recent studies using these new technologies and discuss the utility of transcriptome analysis.

Although evidence has accumulated on the role of plant peptides in the response to external conditions, the number of peptide-encoding genes in the genome is still underestimated. Using tiling arrays, we identified 176 unannotated transcriptionally active regions (TARs) in Arabidopsis thaliana that were induced upon oxidative stress generated by the herbicide paraquat (PQ). These 176 TARs could be translated into 575 putative oxidative stress-induced peptides (OSIPs). A high-throughput functional assay was used in the eukaryotic model organism Saccharomyces cerevisiae allowing us to test for bioactive peptides that increase oxidative stress tolerance. In this way, we identified three OSIPs that, upon overexpression in yeast, resulted in a significant rise in tolerance to hydrogen peroxide (H2O2). For one of these peptides, the decapeptide OSIP108, exogenous application to H2O2-treated yeast also resulted in significantly increased survival. OSIP108 is contained within a pseudogene and is induced in A. thaliana leaves by both the reactive oxygen species-inducer PQ and the necrotrophic fungal pathogen Botrytis cinerea. Moreover, infiltration and overexpression of OSIP108 in A. thaliana leaves resulted in increased tolerance to treatment with PQ. In conclusion, the identification and characterization of OSIP108 confirms the validity of our high-throughput approach, based on tiling array analysis in A. thaliana and functional screening in yeast, to identify bioactive peptides. [ABSTRACT FROM AUTHOR]

Abstract: When the whole human genome sequence was determined by the Human Genome Project, the number of identified genes was fewer than expected. However, recent studies suggest that undiscovered transcripts still exist in the human genome. Furthermore, a new technology, the DNA microarray, which can simultaneously characterize huge amounts of genome sequence data, has become a useful tool for analyzing genetic changes in various diseases. A version of this tool, the tiling DNA microarray, was designed to search all the transcripts of the entire human genome, and provides huge amounts of data, including both exon and intron sequences, by a simple process. Although some previous studies using tiling DNA microarray analysis have indicated that numerous novel transcripts can be found in the human genome, none of them has reported any novel full-length human genes. Here, to find novel genes, we analyzed all the transcripts expressed in normal human prostate cells using this microarray. Because the optimal analytical parameters for using tiling DNA microarray data for this purpose had not been established, we established parameters for extracting the most likely regions for novel transcripts. The three parameters we optimized were the threshold for positive signal intensity, the Max gap, and the Min run, which we set to detect all transcriptional regions that were above the average length of known exons and had a signal intensity in the top 5%. We succeeded in obtaining the full-length sequence of one novel gene, located on chromosome 12q24.13. We named the novel gene “POTAGE”. Its 5841-bp mRNA consists of 26 exons. We detected part of exon 2 in the tiling data analysis. The full-length sequence was then obtained by RT-PCR and RACE. Although the function of POTAGE is unclear, its sequence showed high homology with genes in other species, suggesting it might have an important or essential function. This study demonstrates that the tiling DNA microarray can be useful for identifying novel human genes. [Copyright &y& Elsevier]

Abstract: Most eukaryotes regulate commonly translation by polyadenylation of messenger RNA. RNA sequences without 3′ polyadenylation are usually quickly degraded, thus finely tuning the expression of gene transcripts. Some viruses are known to regulate mRNA levels via polyadenylation. A method is described that utilizes a customized tiling array to determine if any human cytomegalovirus (HCMV) transcripts are subject to regulation via de-polyadenylation and subsequent mRNA decay. The results of this study indicate that the highly expressed intron of large non-coding RNA (lncRNA5) that is not polyadenylated, as documented previously. This differential polyadenylation protocol can also be modified for other tiling array applications (such as ChIP-chip analysis). [Copyright &y& Elsevier]

DNA methylation is an epigenetic modification that plays an important role in many biological processes. To better understand this epigenetic mechanism, genome-wide investigations of DNA methylation can be conducted via tiling array technology. In such DNA methylation profiling studies, statistical methods are employed to estimate the DNA methylation status of each probe represented on the tiling array. Although many of the current methods account for the inherent dependency between neighboring probes, they do not effectively utilize the wealth of information available in genomic annotation databases. Since previous studies indicate that DNA methylation patterns of some organisms differ by genomic element (e.g., gene, intergenic region), such genomic annotation information may be useful in predicting DNA methylation status. In this work, a novel statistical model is proposed within the hidden Markov model framework to incorporate genomic annotation information in the prediction of DNA methylation status. The advantages of incorporating genomic annotation via the proposed method are investigated through a simulation study and real data applications. [ABSTRACT FROM AUTHOR]

Abstract: The analysis of the genetic variability associated to Alu sequences was hampered by the absence of genome-wide methodologies able to efficiently detect new polymorphisms/mutations among these repetitive elements. Here we describe two Alu insertion profiling (AIP) methods based on the hybridization of Alu-flanking genomic fragments on tiling microarrays. Protocols are designed to preferentially detect active Alu subfamilies. We tested AIP methods by analyzing chromosomes 1 and 6 in two genomic samples. In genomic regions covered by array-features, with a sensitivity of 2% (AIP1) −4% (AIP2) and 5% (AIP1) −8% (AIP2) for the old J and S Alu lineages respectively, we obtained a sensitivity of 67% (AIP1) −90% (AIP2) for the young Ya subfamily. Among the loci showing sample-to-sample differences, 5 (AIP1) −8 (AIP2) were associated to known Alu polymorphisms. Moreover, we were able to confirm by PCR and DNA sequencing 4 new intragenic Alu elements, polymorphic in 10 additional individuals. [Copyright &y& Elsevier]

Abstract: Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin®) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level. [Copyright &y& Elsevier]

Background: As a powerful tool in whole genome analysis, tiling array has been widely used in the answering of many genomic questions. Now it could also serve as a capture device for the library preparation in the popular high throughput sequencing experiments. Thus, a flexible and efficient tiling array design approach is still needed and could assist in various types and scales of transcriptomic experiment. Results: In this paper, we address issues and challenges in designing probes suitable for tiling array applications and targeted sequencing. In particular, we define the penalized uniqueness score, which serves as a controlling criterion to eliminate potential cross-hybridization, and a flexible tiling array design pipeline. Unlike BLAST or simple suffix array based methods, computing and using our uniqueness measurement can be more efficient for large scale design and require less memory. The parameters provided could assist in various types of genomic tiling task. In addition, using both commercial array data and experiment data we show, unlike previously claimed, that palindromic sequence exhibiting relatively lower uniqueness. Conclusions: Our proposed penalized uniqueness score could serve as a better indicator for cross hybridization with higher sensitivity and specificity, giving more control of expected array quality. The flexible tiling design algorithm incorporating the penalized uniqueness score was shown to give higher coverage and resolution. The package to calculate the penalized uniqueness score and the described probe selection algorithm are implemented as a Perl program, which is freely available at http://www1.fbn-dummerstorf.de/en/forschung/fbs/fb3/paper/2012- yang-1/OTAD.v1.1.tar.gz. [ABSTRACT FROM AUTHOR]

Microarrays are routine tools for transcript profiling, and genomic tiling arrays such as the Arabidopsis AGRONOMICS1 arrays have been found to be highly suitable for such experiments because changes in genome annotation can be easily integrated at the data analysis level. In a transcript profiling experiment, RNA labeling is a critical step, most often initiated by oligo-dT-primed reverse transcription. Although this has been found to be a robust and reliable method, very long transcripts or non-polyadenylated transcripts might be labeled inefficiently. In this study, we first provide data handling methods to analyze AGRONOMICS1 tiling microarrays based on the TAIR10 genome annotation. Second, we describe methods to easily quantify antisense transcripts on such tiling arrays. Third, we test a random-primed RNA labeling method, and find that on AGRONOMICS1 arrays this method has similar general performance as the conventional oligo-dT-primed method. In contrast to the latter, however, the former works considerably better for long transcripts and for non-polyadenylated transcripts such as found in mitochondria and plastids. We propose that researchers interested in organelle function use the random-primed method to unleash the full potential of genomic tiling arrays. [ABSTRACT FROM AUTHOR]

A complex network of epigenetic factors participates in regulating the monoallelic expression of a small subset of genes (˜1%) in the human genome. This phenomenon goes under the definition of genomic imprinting, a parent-of-origin effect that, when altered during early embryogenesis, may influence fetal development into adulthood. Pertubations in genomic imprinting have been associated with placental and fetal growth restrictions. We analyzed the differential DNA methylation of all known imprinted genes on 10 appropriate-for-gestational-age, clinically normal, placentas and 7 severe intrauterine growth-restricted placentas. Samples were pooled according to the diagnosis and analyzed by methylated DNA immunoprecipitation (MeDIP) on a tiling microarray platform. The distribution of the differentially methylated regions (DMRs) identified in growth-restricted placentas showed a slight tendency toward hypermethylation. Imprinted genes not expressed in placenta showed a unique DMR profile with the fewest hyper- and hypomethylated DMRs. Promoter and CpG island DMRs were sporadic and randomly distributed. The vast majority of DMR identified (˜99%) were mapped in introns, showing no common sequence features. Also, by using the more advanced array data mining softwares, no significant patterns emerged. In contrast, differential methylation showed a highly significant correlation with gene length. Overall these data suggest that differential methylation changes in growth-restricted placentas occur throughout the genomic regions, encompassing genes actively expressed in the placenta. These findings warrant caution in interpreting the significance of genes carrying clustered DMRs because the distribution of DMRs in a gene may be attributed as a function of its length rather than as a specific biological role. [ABSTRACT FROM PUBLISHER]

We have demonstrated that an Arabidopsis serine/arginine rich-like protein, atSR45a, interacts with other splicing factors and its expression is markedly induced by high-light stress, suggesting the involvement of atSR45a in the regulation of stress-responsive alternative splicing. A whole-genome tiling array identified the alternative splicing of genes regulated by atSR45a by comparing gene expression profiles in wild-type and knockout atSR45a (KO-sr45a) plants under high-light stress. The expression levels of genomic regions within 217 genes were significantly altered in the KO-sr45a plants compared with the wild-type plants. Many genes encoded factors involved in signal transduction, cell cycle and DNA processing, protein fate and transcription. A semi-quantitative reverse transcription–PCR (RT–PCR) analysis confirmed changes in the transcript levels and/or alternative splicing efficiency under high-light stress in 18 genes, suggesting that atSR45a affects directly or indirectly not only alternative splicing efficiency but also the transcription of these target genes. Changes in the expression of atSR45a in response to high-light stress temporally correlated with changes in the alternative splicing efficiency and transcript levels of three and one target genes, respectively. Sequencing of the alternatively spliced variants of three target genes showed that atSR45a suppresses the splicing efficiency of intron retention-type alternative splicing events. These findings indicated the importance of atSR45a to the diversification of the transcriptome under high-light stress. [ABSTRACT FROM AUTHOR]

Microarrays first made the analysis of the transcriptome possible, and have produced much important information. Today, however, researchers are increasingly turning to direct high-throughput sequencing - RNA-Seq - which has considerable advantages for examining transcriptome fine structure - for example in the detection of allele-specific expression and splice junctions. In this article, we discuss the relative merits of the two techniques, the inherent biases in each, and whether all of the vast body of array work needs to be revisited using the newer technology. We conclude that microarrays remain useful and accurate tools for measuring expression levels, and RNA-Seq complements and extends microarray measurements. [ABSTRACT FROM AUTHOR]

Background: Changes in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease. Results: In this study Me thylated D NA I mmunoP recipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify T issue and D evelopmental S tage specific D ifferentially M ethylated DNA R egions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array). Conclusions: Our data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation. [ABSTRACT FROM AUTHOR]