Pseudogenes as an alternative source of natural antisense transcripts

<p>Abstract</p> <p>Background</p> <p>Naturally occurring antisense transcripts (NATs) are non-coding RNAs that may regulate the activity of sense transcripts to which they bind because of complementarity. NATs that are not located in the gene they regulate (trans-NATs) have better chances to evolve than cis-NATs, which is evident when the sense strand of the cis-NAT is part of a protein coding gene. However, the generation of a trans-NAT requires the formation of a relatively large region of complementarity to the gene it regulates.</p> <p>Results</p> <p>Pseudogene formation may be one evolutionary mechanism that generates trans-NATs to the parental gene. For example, this could occur if the parental gene is regulated by a cis-NAT that is copied as a trans-NAT in the pseudogene. To support this we identified human pseudogenes with a trans-NAT to the parental gene in their antisense strand by analysis of the database of expressed sequence tags (ESTs). We found that the mutations that appeared in these trans-NATs after the pseudogene formation do not show the flat distribution that would be expected in a non functional transcript. Instead, we found higher similarity to the parental gene in a region nearby the 3' end of the trans-NATs.</p> <p>Conclusions</p> <p>Our results do not imply a functional relation of the trans-NAT arising from pseudogenes over their respective parental genes but add evidence for it and stress the importance of duplication mechanisms of genetic material in the generation of non-coding RNAs. We also provide a plausible explanation for the large transcripts that can be found in the antisense strand of some pseudogenes.</p

Amplification of the Gene Ontology annotation of Affymetrix probe sets

BACKGROUND: The annotations of Affymetrix DNA microarray probe sets with Gene Ontology terms are carefully selected for correctness. This results in very accurate but incomplete annotations which is not always desirable for microarray experiment evaluation. RESULTS: Here we present a protocol to amplify the set of Gene Ontology annotations associated to Affymetrix DNA microarray probe sets using information from related databases. CONCLUSION: Predicted novel annotations and the evidence producing them can be accessed at Probe2GO: . Scripts are available on demand

HetR-dependent and -independent expression of heterocyst-related genes in an Anabaena strain overproducing the NtcA transcription factor

Heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen control transcription factor NtcA and the cell differentiation regulatory protein HetR, with expression of ntcA and hetR being dependent on each other. In this study we constructed strains that constitutively express the ntcA gene leading to high levels of NtcA protein irrespective of the nitrogen source, and we analyzed the elects of such NtcA levels on heterocyst diferentiation. In the NtcA-overproducing strain, heterocyst differentiation, induction of NtcA-dependent heterocyst development genes or operons such as devBCA or the cox2 operon, and NtcA-dependent excision of the 11-kb nifD-intervening element only took place under nitrogen deficiency. Although functional heterocysts were produced in response to nitrogen step-down, the NtcA over-producing strain could not grow diazotrophically. Overexpression of ntcA in a hetR background promoted expression of devBCA in response to ammonium withdrawal and excision of the 11-kb element even in the presence of combined nitrogen. Our results show that some NtcA-dependent heterocyst-related genes can be expressed independently of HetR. Copyright © 2005, American Society for Microbiology. All Rights Reserved.Peer Reviewe

Localized induction of the ntcA regulatory gene in developing heterocysts of Anabaena sp. strain PCC 7120

The ntcA gene encodes an N-control transcriptional regulator in cyanobacteria. In the N2-fixing, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ntcA is an autoregulatory gene that is transcribed from a complex promoter region that includes a constitutive promoter (P 2) and promoters that are induced upon N step-down (P1 and P3). Expression of ntcA was investigated with the use of an ntcA-gfp translational fusion, which was introduced both in the natural ntcA locus and in a heterologous genomic place. Induction of ntcA-gfp took place after N step-down in all the cells of the filament, but at especially high levels in developing heterocysts. Localized induction could be driven independently by P3 and P1. Copyright © 2006, American Society for Microbiology. All Rights Reserved.Peer Reviewe

The pseudogenes of Mycobacterium leprae reveal the functional relevance of gene order within operons

Almost 50 years following the discovery of the prokaryotic operon, the functional relevance of gene order within operons remains unclear. In this work, we take advantage of the eroded genome of Mycobacterium leprae to add evidence supporting the notion that functionally less important genes have a tendency to be located at the end of its operons. M. leprae’s genome includes 1133 pseudogenes and 1614 protein-coding genes and can be compared with the close genome of M. tuberculosis. Assuming M. leprae’s pseudogenes to represent dispensable genes, we have studied the position of these pseudogenes in the operons of M. leprae and of their orthologs in M. tuberculosis. We observed that both tend to be located in the 3′ (downstream) half of the operon (P-values of 0.03 and 0.18, respectively). Analysis of pseudogenes in all available prokaryotic genomes confirms this trend (P-value of 7.1 × 10−7). In a complementary analysis, we found a significant tendency for essential genes to be located at the 5′ (upstream) half of the operon (P-value of 0.006). Our work provides an indication that, in prokarya, functionally less important genes have a tendency to be located at the end of operons, while more relevant genes tend to be located toward operon starts

Role of two NtcA-binding sites in the complex ntcA gene promoter of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that fixes N2 in specialized cells called heterocysts, which differentiate from vegetative cells in a process that requires the nitrogen control transcription factor NtcA. 2-Oxoglutarate-stimulated binding of purified NtcA to wild-type and modified versions of the ntcA gene promoter from Anabaena sp. was analyzed by mobility shift and DNase I footprinting assays, and the role of NtcA-binding sites in the expression of the ntcA gene during heterocyst differentiation was studied in vivo by using an ntcA-gfp translational fusion and primer extension analysis. Mutation of neither of the two identified NtcA-binding sites eliminated localized expression of ntcA in proheterocysts, but mutation of both sites led to very low, nonlocalized expression. Copyright © 2008, American Society for Microbiology. All Rights Reserved.Peer Reviewe

The coxBAC Operon Encodes a Cytochrome c Oxidase Required for Heterotrophic Growth in the Cyanobacterium Anabaena variabilis Strain ATCC 29413

Three genes, coxB, coxA, and coxC, found in a clone from a gene library of the cyanobacterium Anabaena variabilis strain ATCC 29413, were identified by hybridization with an oligonucleotide specific for aa3-type cytochrome c oxidases. Deletion of these genes from the genome of A. variabilis strain ATCC 29413 FD yielded strain CSW1, which displayed no chemoheterotrophic growth and an impaired cytochrome c oxidase activity. Photoautotrophic growth of CSW1, however, was unchanged, even with dinitrogen as the nitrogen source. A higher cytochrome c oxidase activity was detected in membrane preparations from dinitrogen-grown CSW1 than from nitrate-grown CSW1, but comparable activities of respiratory oxygen uptake were found in the wild type and in CSW1. Our data indicate that the identified cox gene cluster is essential for fructose-dependent growth in the dark, but not for growth on dinitrogen, and that other terminal respiratory oxidases are expressed in this cyanobacterium. Transcription analysis showed that coxBAC constitutes an operon which is expressed from two transcriptional start points. The use of one of them was stimulated by fructose.Peer Reviewe

ChIP on SNP-chip for genome-wide analysis of human histone H4 hyperacetylation

<p>Abstract</p> <p>Background</p> <p>SNP microarrays are designed to genotype Single Nucleotide Polymorphisms (SNPs). These microarrays report hybridization of DNA fragments and therefore can be used for the purpose of detecting genomic fragments.</p> <p>Results</p> <p>Here, we demonstrate that a SNP microarray can be effectively used in this way to perform chromatin immunoprecipitation (ChIP) on chip as an alternative to tiling microarrays. We illustrate this novel application by mapping whole genome histone H4 hyperacetylation in human myoblasts and myotubes. We detect clusters of hyperacetylated histone H4, often spanning across up to 300 kilobases of genomic sequence. Using complementary genome-wide analyses of gene expression by DNA microarray we demonstrate that these clusters of hyperacetylated histone H4 tend to be associated with expressed genes.</p> <p>Conclusion</p> <p>The use of a SNP array for a ChIP-on-chip application (ChIP on SNP-chip) will be of great value to laboratories whose interest is the determination of general rules regarding the relationship of specific chromatin modifications to transcriptional status throughout the genome and to examine the asymmetric modification of chromatin at heterozygous loci.</p

Recent developments in StemBase: a tool to study gene expression in human and murine stem cells

<p>Abstract</p> <p>Background</p> <p>Currently one of the largest online repositories for human and mouse stem cell gene expression data, StemBase was first designed as a simple web-interface to DNA microarray data generated by the Canadian Stem Cell Network to facilitate the discovery of gene functions relevant to stem cell control and differentiation.</p> <p>Findings</p> <p>Since its creation, StemBase has grown in both size and scope into a system with analysis tools that examine either the whole database at once, or slices of data, based on tissue type, cell type or gene of interest. As of September 1, 2008, StemBase contains gene expression data (microarray and Serial Analysis of Gene Expression) from 210 stem cell samples in 60 different experiments.</p> <p>Conclusion</p> <p>StemBase can be used to study gene expression in human and murine stem cells and is available at <url>http://www.stembase.ca</url>.</p

Gene function in early mouse embryonic stem cell differentiation

BACKGROUND: Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC) differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5) undergoing undirected differentiation into embryoid bodies (EBs) over a period of two weeks. RESULTS: We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1), our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2) that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. CONCLUSION: Our analysis profiles for the first time gene expression at a very early stage of mESC differentiation, and identifies a functional and phylogenetic signature for the genes involved. The data generated constitute a valuable resource for further studies. All DNA microarray data used in this study are available in the StemBase database of stem cell gene expression data [1] and in the NCBI's GEO database