Putative Promoter Motifs Research Articles

RNA Sequencing of Medusavirus Suggests Remodeling of the Host Nuclear Environment at an Early Infection Stage.

ABSTRACTViruses of the phylum Nucleocytoviricota, or nucleo-cytoplasmic large DNA viruses (NCLDVs), undergo a cytoplasmic or nucleo-cytoplasmic cycle, the latter of which involves both nuclear and cytoplasmic compartments to proceed viral replication. Medusavirus, a recently isolated NCLDV, has a nucleo-cytoplasmic replication cycle in amoebas during which the host nuclear membrane apparently remains intact, a unique feature among amoeba-infecting NCLDVs. The medusavirus genome lacks most transcription genes but encodes a full set of histone genes. To investigate its infection strategy, we performed a time course RNA sequencing (RNA-seq) experiment. All viral genes were transcribed and classified into five temporal expression clusters. The immediate early genes (cluster 1, 42 genes) were mostly (83%) of unknown functions, frequently (95%) associated with a palindromic promoter-like motif, and often (45%) encoded putative nucleus-localized proteins. These results suggest massive reshaping of the host nuclear environment by viral proteins at an early stage of infection. Genes in other expression clusters (clusters 2 to 5) were assigned to various functional categories. The virally encoded core histone genes were in cluster 3, whereas the viral linker histone H1 gene was in cluster 1, suggesting they have distinct roles during the course of the virus infection. The transcriptional profile of the host Acanthamoeba castellanii genes was greatly altered postinfection. Several encystment-related host genes showed increased representation levels at 48 h postinfection, which is consistent with the previously reported amoeba encystment upon medusavirus infection.IMPORTANCE Medusavirus is an amoeba-infecting giant virus that was isolated from a hot spring in Japan. It belongs to the proposed family “Medusaviridae” in the phylum Nucleocytoviricota. Unlike other amoeba-infecting giant viruses, medusavirus initiates its DNA replication in the host nucleus without disrupting the nuclear membrane. Our RNA sequencing (RNA-seq) analysis of its infection course uncovered ordered viral gene expression profiles. We identified temporal expression clusters of viral genes and associated putative promoter motifs. The subcellular localization prediction showed a clear spatiotemporal correlation between gene expression timing and localization of the encoded proteins. Notably, the immediate early expression cluster was enriched in genes targeting the nucleus, suggesting the priority of remodeling the host intranuclear environment during infection. The transcriptional profile of amoeba genes was greatly altered postinfection.

Analysis of a Marseillevirus Transcriptome Reveals Temporal Gene Expression Profile and Host Transcriptional Shift.

Marseilleviruses comprise a family of large double-stranded DNA viruses belonging to the proposed order “Megavirales.” These viruses have a circular genome of ∼370 kbp, coding hundreds of genes. Over a half of their genes are associated with AT-rich putative promoter motifs, which have been demonstrated to be important for gene regulation. However, the transcriptional profile of Marseilleviruses is currently unknown. Here we used RNA sequencing technology to get a general transcriptional profile of Marseilleviruses. Eight million 75-bp-long nucleotide sequences were robustly mapped to all 457 genes initially predicted for Marseillevirus isolate T19, the prototype strain of the family, and we were able to assemble 359 viral contigs using a genome-guided approach with stringent parameters. These reads were differentially mapped to the genes according to the replicative cycle time point from which they were obtained. Cluster analysis indicated the existence of three main temporal categories of gene expression, early, intermediate and late, which were validated by quantitative reverse transcription polymerase chain reaction assays targeting several genes. Genes belonging to different functional groups exhibited distinct expression levels throughout the infection cycle. We observed that the previously predicted promoter motif, AAATATTT, as well as new predicted motifs, were not specifically related to any of the temporal or functional classes of genes, suggesting that other components are involved in temporally regulating virus transcription. Moreover, the host transcription machinery is heavily altered, and many genes are down regulated, including those related to translation process. This study provides an overview of the transcriptional landscape of Marseilleviruses.

Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

ABSTRACTMicro (mi)RNAs regulate gene expression in many eukaryotic organisms where they control diverse biological processes. Their biogenesis, from primary transcripts to mature miRNAs, have been extensively characterized in animals and plants, showing distinct differences between these phylogenetically distant groups of organisms. However, comparably little is known about miRNA biogenesis in organisms whose evolutionary position is placed in between plants and animals and/or in unicellular organisms. Here, we investigate miRNA maturation in the unicellular amoeba Dictyostelium discoideum, belonging to Amoebozoa, which branched out after plants but before animals. High-throughput sequencing of small RNAs and poly(A)-selected RNAs demonstrated that the Dicer-like protein DrnB is required, and essentially specific, for global miRNA maturation in D. discoideum. Our RNA-seq data also showed that longer miRNA transcripts, generally preceded by a T-rich putative promoter motif, accumulate in a drnB knock-out strain. For two model miRNAs we defined the transcriptional start sites (TSSs) of primary (pri)-miRNAs and showed that they carry the RNA polymerase II specific m7G-cap. The generation of the 3ʹ-ends of these pri-miRNAs differs, with pri-mir-1177 reading into the downstream gene, and pri-mir-1176 displaying a distinct end. This 3´-end is processed to shorter intermediates, stabilized in DrnB-depleted cells, of which some carry a short oligo(A)-tail. Furthermore, we identified 10 new miRNAs, all DrnB dependent and developmentally regulated. Thus, the miRNA machinery in D. discoideum shares features with both plants and animals, which is in agreement with its evolutionary position and perhaps also an adaptation to its complex lifestyle: unicellular growth and multicellular development.

Putative Promoter Motif Analyses Reinforce the Evolutionary Relationships Among Faustoviruses, Kaumoebavirus, and Asfarvirus.

Putative promoter motifs have been described in viruses belonging to the nucleocytoplasmic large DNA viruses (NCLDVs) group; however, few studies have been conducted to search for promoter sequences in newly discovered amoebal giant viruses. Faustovirus and kaumoebavirus are two Asfarviridae-related giant viruses belonging to the NCLDVs group. The phylogenetic relationships among these viruses led us to investigate if the promoter regions previously identified in the asfarvirus genome could be shared by its amoebal virus relatives. Previous studies demonstrated the role of A/T-rich motifs as promoters of asfarvirus. In this study, we reinforce the importance of A/T rich motifs in asfarvirus and show that the TATTT and TATATA motifs are also shared in abundance by faustovirus and kaumoebavirus. Here, we demonstrate that TATTT and TATATA are mostly present in faustovirus and kaumoebavirus genomic intergenic regions (IRs) and that they are widely distributed at 0 to -100 bp upstream to the start codons. We observed that putative promoter motifs are present as one to dozens of repetitions in IRs of faustovirus, kaumoebavirus, and asfarvirus, which is similar to that described previously for marseilleviruses. Furthermore, the motifs were found in most of the upstream regions of the core genes of faustovirus, kaumoebavirus, and asfarvirus, which suggests that the motifs could already be present in the ancestor of these viruses before the irradiation of this group. Our work provides an in-depth analysis of the putative promoter motifs present in asfarvirus, kaumoebavirus, and faustovirus, which reinforces the relationship among these viruses.

The Investigation of Promoter Sequences of Marseilleviruses Highlights a Remarkable Abundance of the AAATATTT Motif in Intergenic Regions.

Viruses display a wide range of genomic profiles and, consequently, a variety of gene expression strategies. Specific sequences associated with transcriptional processes have been described in viruses, and putative promoter motifs have been elucidated for some nucleocytoplasmic large DNA viruses (NCLDV). Among NCLDV, the Marseilleviridae is a well-recognized family because of its genomic mosaicism. The marseilleviruses have an ability to incorporate foreign genes, especially from sympatric organisms inhabiting Acanthamoeba, its main known host. Here, we identified for the first time an eight-nucleotide A/T-rich promoter sequence (AAATATTT) associated with 55% of marseillevirus genes that is conserved in all marseilleviruses lineages, a higher level of conservation than that of any giant virus described to date. We instigated our prediction about the promoter motif by biological assays and by evaluating how single mutations in this octamer can impact gene expression. The investigation of sequences that regulate the expression of genes relative to lateral transfer revealed that the promoter motifs do not appear to be incorporated by marseilleviruses from donor organisms. Indeed, analyses of the intergenic regions that regulate lateral gene transfer-related genes have revealed an independent origin of the marseillevirus intergenic regions that does not match gene-donor organisms. About 50% of AAATATTT motifs spread throughout intergenic regions of the marseilleviruses are present as multiple copies. We believe that such multiple motifs are associated with increased expression of a given gene or are related to incorporation of foreign genes into the mosaic genome of marseilleviruses.IMPORTANCE The marseilleviruses draw attention because of the peculiar features of their genomes; however, little is known about their gene expression patterns or the factors that regulate those expression patterns. The limited published research on the expression patterns of the marseilleviruses and their unique genomes has led us to study the promoter motif sequences in the intergenic regions of the marseilleviruses. This work is the first to analyze promoter sequences in the genomes of the marseilleviruses. We also suggest a strong capacity to acquire foreign genes and to express those genes mediated by multiple copies of the promoter motifs available in intergenic regions. These findings contribute to an understanding of genomic expansion and plasticity observed in these giant viruses.

Comparative Genomics Reveals Two Major Bouts of Gene Retroposition Coinciding with Crucial Periods of Symbiodinium Evolution.

Gene retroposition is an important mechanism of genome evolution but the role it plays in dinoflagellates, a critical player in marine ecosystems, is not known. Until recently, when the genomes of two coral-symbiotic dinoflagellate genomes, Symbiodinium kawagutii and S. minutum, were released, it has not been possible to systematically study these retrogenes. Here we examine the abundant retrogenes (∼23% of the total genes) in these species. The hallmark of retrogenes in the genome is the presence of DCCGTAGCCATTTTGGCTCAAG, a spliced leader (DinoSL) constitutively trans-spliced to the 5′-end of all nucleus-encoded mRNAs. Although the retrogenes have often lost part of the 22-nt DinoSL, the putative promoter motif from the DinoSL, TTT(T/G), is consistently retained in the upstream region of these genes, providing an explanation for the high survival rate of retrogenes in dinoflagellates. Our analysis of DinoSL sequence divergence revealed two major bursts of retroposition in the evolutionary history of Symbiodinium, occurring at ∼60 and ∼6 Ma. Reconstruction of the evolutionary trajectory of the Symbiodinium genomes mapped these 2 times to the origin and rapid radiation of this dinoflagellate lineage, respectively. GO analysis revealed differential functional enrichment of the retrogenes between the two episodes, with a broad impact on transport in the first bout and more localized influence on symbiosis-related processes such as cell adhesion in the second bout. This study provides the first evidence of large-scale retroposition as a major mechanism of genome evolution for any organism and sheds light on evolution of coral symbiosis.

Proteomics of Sphingobium indicum B90A for a deeper understanding of hexachlorocyclohexane (HCH) bioremediation.

Genome wide expression profiling of Sphingobium indicum B90A revealed induction of lin genes, linA and linB, involved in dechlorination of hexachlorocyclohexane (HCH), in the presence of all four isomers of HCH. Supporting proteomics data, the qPCR and promoter assay showed upregulation of linA transcription in the presence of HCH isomers. Analysis of the upstream region of the linA gene revealed the existence of the GntR binding site overlapping the -10 hexamer of the putative promoter motif. As GntR is a known transcription repressor its dissociation from the linA promoter is expected to induce lin genes in the presence of HCH isomers. Comparison of in situ and in-culture proteomics indicated expression lin genes at the dumpsite, an indication for the in situ HCH degradation.

Identification and analysis of putative promoter motifs in bovine herpes virus.

The purpose of this study is to identify and analyse the putative promoter motifs in the bovine herpes virus which causes several diseases in cattle worldwide including bovine mastitis with large economic impact on dairy industry. Bovine mastitis caused due to virus is often neglected as bacterial infections are held mainly responsible for the disease. Therefore, in this in silico investigation with all the existing experimental data a total of 147 promoter were identified along with their sequences from three genome viz bovine herpes virus 1 (BHV), bovine herpes virus 4 and bovine herpes virus 5, out of which 39 promoters were from bovine herpes virus 4 (BHV 4), 95 from BHV1 and 13 from BHV5 and it was observed that BHV1 and BHV5 have a close evolutionary history. However, they belong to the same subfamily and size of the genome and GC% of BHV1 and BHV5 was almost equal and very high compare to that of BHV4. This analysis may help in designing the live attenuated vaccine against BHV causing bovine mastitis that reduces the incidence of bovine mastitis. Identification of promoters may also help in designing of expression vectors which help in better understanding of the regulation of gene expression. In the era of large genomics and proteomics prediction of promoters in the whole genome is crucial for the advancement of drug discovery and gene therapy.

Identification and analysis of putative promoter motifs in Flavivirus genome

The genus Flavivirus comprises medically significant pathogenic virus; causing several infections in humans worldwide. Flavivirus genomes are 10-11 kb approximately and encode both structural and non structural region. The non structural region plays fundamental role in the stability, regulation and cell cycle of virus. The complete genomes of 26 Flavivirus were used for identification of promoter motifs through in silico approaches. The promoter sequences were encoded in merely 16 viruses and 10 viruses could not encode it. All these in silico identified promoter motifs was confirmed and verified with the known experimental data. This analysis suggests that presence of promoter may play a crucial role in the pattern of gene expression, regulation networks, cell specificity and development. It may also be useful for designing efficient expression vector and target specific delivery system in the gene therapy.

Evolution of the “OR37” Subfamily of Olfactory Receptors: A Cross-Species Comparison

Genes encoding the olfactory receptors of the "OR37" subfamily of the mouse are characterized by special features including a clustered expression pattern, assembly in two distinct gene clusters, and highly conserved putative promoter motifs. Mining the rat and dog databases revealed that these two species possess highly conserved clusters of OR37 genes at two syntenic genomic loci. In a prototherian mammal, the platypus (Ornithorhynchus anatinus), none of the characteristic OR37 genes were found. Examination of a metatherian mammal, the gray short-tailed opossum (Monodelphis domestica) revealed seven canonical OR37 genes, all phylogenetically related to cluster II genes and also organized similar to cluster II of eutherian species. In addition, their 5' upstream regions comprised sequence motifs related to the putative regulatory sequences of cluster II genes. Typical cluster I OR37 genes were identified only in the eutherian mammals examined, including the evolutionary ancient anteater, wherein OR37 genes related to both clusters were present. Together, these results reveal novel information concerning the phylogenetic origin and important evolutionary steps of the mammalian-specific OR37 olfactory receptor family.

Differentiation-dependent activation of the human intestinal alkaline phosphatase promoter by HNF-4 in intestinal cells

The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the enterocytes, we have conducted a computer-assisted cis-element search of the proximal human ALPI promoter sequence. A putative recognition site for the transcription factor hepatocyte nuclear factor (HNF)-4 was predicted at the positions from -94 to -82 in relation to the translational start site. The ability of HNF-4alpha to stimulate the expression from the ALPI promoter was investigated in the nonintestinal Hela cell line. Cotransfection with an HNF-4alpha expression vector demonstrated a direct activation of the ALPI promoter through this -94 to -82 element. EMSA showed that HNF-4alpha from nuclear extracts of differentiated intestinal epithelial cells (Caco-2) bound with high affinity to the predicted HNF-4 binding site. A 521 bp promoter fragment containing the HNF-4 binding site demonstrated a differentiation-dependent increase in promoter activity in Caco-2 cells. The presence of the HNF-4 binding site was necessary for this increase to occur.

New insertion sequence elements in the upstream region of cfiA in imipenem-resistant Bacteroides fragilis strains.

The 747-bp cfiA gene, which encodes a metallo-beta-lactamase, and the regions flanking cfiA in six imipenem-resistant and four imipenem-susceptible Bacteroides fragilis strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the cfiA genes (designated cfiA(1) to cfiA(10)) of all 10 strains tested varied from that of the standard cfiA gene from B. fragilis TAL2480. However, putative proteins encoded by the cfiA variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that cfiA variants have the capability of producing metallo-beta-lactamases with full catalytic activities. PCR assay indicated that six metallo-beta-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of cfiA. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of cfiA revealed that there were five new kinds of insertion sequence (IS) elements (designated IS612, IS613, IS614, IS615, and IS616, with a size range of 1,594 to 1,691 bp), of which only IS616 was found to be almost identical to IS1188, one of the IS elements previously identified in the upstream region of cfiA. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of cfiA. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS612, IS613, IS614, and IS615 belong to the IS4 family, which includes IS942, previously found in some drug-resistant B. fragilis strains, but that IS616 belongs to the IS1380 family. All the IS elements appear to have putative promoter motif sequences (the -7 region's TAnnTTTG motif and the -33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of cfiA upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the cfiA gene.

A plasmid vector for isolation of strong promoters in Escherichia coli.

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.

A plasmid vector for isolation of strong promoters in Escherichia coli

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.

Transcription initiation and RNA processing in the mitochondria of the red alga Chondrus crispus: convergence in the evolution of transcription mechanisms in mitochondria

The mitochondrial DNA (mt DNA) of the red alga Chondrus crispus is shown to be transcribed into two large RNA molecules. These primary transcripts are cleaved once, at the level of a tRNA, then the resulting products are processed via multiple maturation events into either mono- or poly-cistronic RNAs. Transcripts were detected for all genes and open reading frames, except for rps11 and orf172. For both transcription units the initiation of transcription was mapped by in vitro RNA capping and primer extension experiments within inverse repeated sequences at the north pole of the molecule. Consistent with primer extension mapping, putative promoter motifs sharing significant similarities with both chicken and Xenopus mitochondrial promoters were found in the C. crispus mitochondrial genome. Altogether C. crispus mitochondrial DNA appears to be transcribed as animal mtDNA is, suggesting that transcription mechanisms in mitochondria are dependent on the overall organization of the mitochondrial genome irrespective of the eukaryotic phylogeny.