Conserved Sequence Blocks Research Articles

The First Complete Mitochondrial Genome of the Flathead Cociella crocodilus (Scorpaeniformes: Platycephalidae) and the Phylogenetic Relationships within Scorpaeniformes Based on Whole Mitogenomes.

Complete mitochondrial genomes (mitogenomes) are important molecular markers for understanding the phylogenetics of various species. Although recent studies on the mitogenomes of the Scorpaeniformes species have been greatly advanced, information regarding molecular studies and the taxonomic localization of Platycephalidae is still sparse. To further analyze the phylogeny of Platycephalidae, we sequenced the complete mitogenome of Cociella crocodilus of the Platycephalidae family within Scorpaeniformes for the first time. The mitogenome was 17,314 bp in length, contained two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), 13 protein-coding genes (PCGs), and two typical noncoding control regions (the control region (CR) and origin of the light strand (OL)). All PCGs used standard initiation codons ATG, apart from cox1. The majority of the tRNA genes could be folded into cloverleaf secondary structures, whereas the secondary structure of tRNASer (AGN) lacked a dihydrouridine (DHU) stem. The CR contained several conserved sequence blocks (CSBs) and eight tandem repeats. In addition, the phylogenetic relationship based on the concatenated nucleotides sequences of 13 PCGs indicated that the Platycephalidae species are relatively basal in the phylogenetic relationships of Scorpaeniformes. Our results may not only advance the origin and the evolution of Scorpaeniformes, but also provide information for the genetic evolution and taxonomy of the teleostean species.

Complete mitochondrial genome of yellowback seabream, Dentex hypselosomus and phylogenetic analysis of the family Sparidae

The complete mitogenome sequence of Dentex hypselosomus was amplified by designing 15 primer pairs. The circle genome was 16,618 bp in size and the overall base composition was 27.04% of A, 26.38% of T, 17.08% of G, and 29.50 of C, with significant anti-G bias. The complete mitogenome of D. hypselosomus encodes 37 canonical mitochondrial genes, two non-coding regions, an L-strand replication region (OL), and a control region (D-loop). The D-loop contained termination sequence domain (TAS), central conserved domains (CSB-F, CSB-E, CSB-D, CSB-C, and CSB-A), and conserved sequence blocks (CSB-1, CSB-2, and CSB-3). Phylogenetic analysis of nine sparid species well supported the phylogenetic position of D. hypselosomus and revealed the phylogenetic relationship of the family Sparidae at the level of mitochondrial genomes.

The second complete mitogenome of Scolopsis ciliata (Perciformes, Nemipteridae) to analyze control region structure and intraspecific variation

In this study, we reported the complete mitogenome of Scolopsis ciliata sampled from the Beibu Gulf, South China Sea. It was 16,734 bp in length and contained 37 canonical mitochondrial genes and 2 non-coding regions. Fifty-one single nucleotide polymorphisms and five insertion and deletions were detected between two S. ciliata mitogenomes, suggesting a high level of intraspecific genetic variation in the species. The control region contained termination associated sequence domain (TAS), central conserved domain (CSB-F, CSB-E, CSB-D, CSB-C, and CSB-A), and conserved sequence block domain (CSB-1, CSB-2, and CSB-3). The phylogenetic analysis of 21 complete mitogenome sequences well supports the phylogenetic position of S. ciliata and reveals the phylogenetic relationship of the genus Scolopsis.

The second complete mitogenome of Nemipterus virgatus to dissect control region structure and phylogenetic problem of the superfamily Sparoidea (Teleostei, Perciformes)

In this study, we determined the complete mitogenome of Nemipterus virgatus of which the length was 17,073 bp, including 37 canonical mitochondrial genes and 2 non-coding regions. The control region contained termination associated sequence domain (TAS), central conserved domain (CSB-F, CSB-E, CSB-D, CSB-C, and CSB-A), conserved sequence block domain (CSB-1, CSB-2, and CSB-3), and tandem repeat sequence domain (TTD). Nine single nucleotide polymorphisms and three insertion of tandem repeat sequence (each length in 28 bp) were detected between two N. virgatus mitogenomes. The phylogenetic analysis showed that the families Nemipteridae, Sparidae, Centracanthidae, and Lethrinidae did not gather into a monophyly of superfamily Sparoidea in the neighbor-joining tree.

The complete mitochondrial genome of Rhynchocypris oxycephalus (Teleostei: Cyprinidae) and its phylogenetic implications.

Rhynchocypris oxycephalus (Teleostei: Cyprinidae) is a typical small cold water fish, which is distributed widely and mainly inhabits in East Asia. Here, we sequenced and determined the complete mitochondrial genome of R. oxycephalus and studied its phylogenetic implication. R. oxycephalus mitogenome is 16,609 bp in length (GenBank accession no.: MH885043), and it contains 13 protein‐coding genes (PCGs), two rRNA genes, 22 tRNA genes, and two noncoding regions (the control region and the putative origin of light‐strand replication). 12 PCGs started with ATG, while COI used GTG as the start codon. The secondary structure of tRNA‐Ser (AGN) lacks the dihydrouracil (DHU) arm. The control region is 943bp in length, with a termination‐associated sequence, six conserved sequence blocks (CSB‐1, CSB‐2, CSB‐3, CSB‐D, CSB‐E, CSB‐F), and a repetitive sequence. Phylogenetic analysis was performed with maximum likelihood and Bayesian methods based on the concatenated nucleotide sequence of 13 PCGs and the complete sequence without control region, and the result revealed that the relationship between R. oxycephalus and R. percnurus is closest, while the relationship with R. kumgangensis is farthest. The genus Rhynchocypris is revealed as a polyphyletic group, and R. kumgangensis had distant relationship with other Rhynchocypris species. In addition, COI and ND2 genes are considered as the fittest DNA barcoding gene in genus Rhynchocypris. This work provides additional molecular information for studying R. oxycephalus conservation genetics and evolutionary relationships.

Corrigendum: Initiation of mtDNA transcription is followed by pausing, and diverges across human cell types and during evolution.

Mitochondrial DNA (mtDNA) genes are long known to be cotranscribed in polycistrones, yet it remains impossible to study nascent mtDNA transcripts quantitatively in vivo using existing tools. To this end, we used deep sequencing (GRO-seq and PRO-seq) and analyzed nascent mtDNA-encoded RNA transcripts in diverse human cell lines and metazoan organisms. Surprisingly, accurate detection of human mtDNA transcription initiation sites (TISs) in the heavy and light strands revealed a novel conserved transcription pausing site near the light-strand TIS. This pausing site correlated with the presence of a bacterial pausing sequence motif, with reduced SNP density, and with a DNase footprinting signal in all tested cells. Its location within conserved sequence block 3 (CSBIII), just upstream of the known transcription-replication transition point, suggests involvement in such transition. Analysis of nonhuman organisms enabled de novo mtDNA sequence assembly, as well as detection of previously unknown mtDNA TIS, pausing, and transcription termination sites with unprecedented accuracy. Whereas mammals (Pan troglodytes, Macaca mulatta, Rattus norvegicus, and Mus musculus) showed a human-like mtDNA transcription pattern, the invertebrate pattern (Drosophila melanogaster and Caenorhabditis elegans) profoundly diverged. Our approach paves the path toward in vivo, quantitative, reference sequence-free analysis of mtDNA transcription in all eukaryotes.

The Complete Mitochondrial Genome of Terapon jarbua (Centrarchiformes: Terapontidae) and Comparative Analysis of the Control Region among Eight Centrarchiformes Species

In this article, we determined and described the complete mitochondrial genome of Terapon jarbua, which is 16 664 bp in length, containing 13 protein-coding genes, two rRNA genes, 22 tRNA genes, one origin of replication on the light-strand (OL), and a putative control region (CR). The gene order and the base composition of T. jarbua mitogenome are similar to that of most other vertebrates. The overall base composition is 27.5% A, 26.0% T, 29.9% C, 16.6% G, respectively, with a slight AT bias (53.5%). All the protein-coding genes use the initiation codon ATG except COI use GTG. Most of them have TAA or TAG as the stop codon, except COII and ND4 use AGA, COIII and Cyt b use an incomplete stop codon TA and T, respectively. In order to further explore the structure of CR in Centrarchiformes, eight CR sequences from representative species were compared. Like that of other teleosts, the CR of the Centrarchiformes is also partitioned into three domains, including the termination associated sequence (TAS), the central conserved sequence blocks (CSB-E, D, B and A), and the conserved sequence blocks (CSB-2 and 3). We hope these results will provide consensus sequences of the conserved units of the CR in Centrarchiformes genomes, as well as useful molecular information for phylogenetic studies of Centrarchiformes.

The complete mitochondrial genome of Coilia brachygnathus (Clupeiformes: Engraulidae: Coilinae) from Huaihe river

In the present study, the complete mitochondrial genome (mitogenome) sequence of Coilia brachygnathus collected from Huaihe river, was determined by long PCR and primer walking methods. Similar to other fishes, the mitogenome is a circular molecule 16,896 bp in length and contains 13 protein-coding genes (PCGs), 2 rRNA genes, 22 transfer RNA genes, and a control region (CR). In addition, the extended termination associated sequence domain (ETAS), the Central conserved domain (CSB-F, CSB-E, and CSB-D) and the Conserved sequence block (CSB1, CSB2, and CSB3) were identified in the control region. Phylogenetic analysis showed that C. brachygnathus first clustered together with C. nasus among Coilia fishes and formed a monophyly. The results will helpful for further understanding classification, phylogenetics, and biogeography of the Coilia fishes.

Sequencing and analysis of the complete mitochondrial genome of the Eurasian least shrew (Sorex minutissimus) from China

The complete mitogenome sequence of the Eurasian least shrew (Sorex minutissimus) was determined using long PCR. The genome was 17,061 bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, 1 origin of L strand replication and 1 control region. The overall base composition of the heavy strand is A (32.6%), C (24.7%), T (29.1%) and G (13.6%). The base compositions present clearly the A–T skew, which is most obviously in the control region and protein-coding genes. The extended termination-associated sequence domain, the central conserved domain and the conserved sequence block domain are defined in the mitochondrial genome control region of the Eurasian least shrew. Mitochondrial genome analyses based on MP, ML, NJ and Bayesian analyses yielded identical phylogenetic trees. The ten Sorex species formed a monophyletic group with the high bootstrap value (100%) in all examinations.

The complete mitochondrial genome of Atrobucca nibe (Perciformes: Sciaenidae)

In this study, the complete mitochondrial genome (mitogenome) sequence of Atrobucca nibe has been determined by polymerase chain reaction and primer walking methods. The mitogenome is a circular molecule of 16,504 bp in length, including the typical structure of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 non-coding regions (control region and L-strand replication origin), the gene contents of which are identical to those observed in most bony fishes. Within the control region, we identified the termination-associated sequence domain (TAS) and the conserved sequence block domain (CSB-F, CSB-E, CSB-D, CSB-C, CSB-B, CSB-A, CSB-1, CSB-2, CSB-3).

Sequencing and analysis of the complete mitochondrial genome of the Siberian large-toothed shrew (Sorex daphaenodon) from China

The complete mitogenome sequence of the Siberian large-toothed shrew (Sorex daphaenodon) was determined using long PCR. The genome was 17,407 bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 23 transfer RNA genes, 1 origin of L strand replication, and 1 control region. The overall base composition of the heavy strand is A (33.1%), C (24.7%), T (29.0%), and G (13.2%). The base compositions present clearly the A–T skew, which is most obvious in the control region and protein-coding genes. The extended termination-associated sequence domain, the central conserved domain, and the conserved sequence block domain are defined in the mitochondrial genome control region of the Siberian large-toothed shrew. Mitochondrial genome analyses based on MP, ML, NJ, and Bayesian analyses yielded identical phylogenetic trees. The 11 Sorex species formed a monophyletic group with the high bootstrap value (100%) in all examinations.

Exploring new features of α-amylases from different source organisms by an in silico approach

A total of 78 full-length protein sequences of α-amylase from different source organisms were subjected to phylogenetic analysis, multiple sequence alignment (MSA), motif search and physiochemical properties. The phylogenetic tree was built using the maximum likelihood method in molecular evolutionary genetics analysis (MEGA) software and was pointed out in two major clusters. One of the clusters included plants and animals, whereas the other one contained fungi, archaea and bacteria. Furthermore, Firmicutes and Proteobacteria are bacterial phylum that placed in the same evolutionary cluster with plants and animals. The deviations from expected clusters were explained by motif analysis. MSA declared three conserved sequence blocks, 505-527, 725-745 and 1010-1030, that were present in all studied species. Moreover, it provided information about highly conserved residues at which three glycine and one aspartic acid residues were conserved. Motif analysis with multiple EM for the motif elicitation server revealed that Motif 4 'HDTGSTQRHWPFPSDHVMQGYAYILTHPGIPCIFYDHFFDW', Motif 6 'EGAGGPSTAFDFTTKGILQEAVKGELWRLRDPQGKPPGMIGWWPERAVTF' and Motif 11 'EQIVKLIAIRKRNGIHSRSSIRILEAEGDLYVAMIDEK VCMKIG' were present only in plants. Pearson correlation analysis to clarify relationships among different physiochemical properties showed a direct correlation between average hydropathy (GRAVY) and the aliphatic index and a reverse correlation between GRAVY and pI and instability indexes.

The complete mitochondrial genome of Pampus minor (Liu & Li, 1998) (Perciformes: Stromateidae)

In this study, the complete mitochondrial genome sequence of Pampus minor has been determined by long polymerase chain reaction and primer walking methods. The mitochondrial genome is a circular molecule of 16,690 bp in length, including the typical structure of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and two non-coding regions (L-strand replication origin and control region), the gene contents of which are identical to those observed in most bony fishes. The overall base composition of the heavy strand shows 29.9% of T, 24.7% of C, 30.6% of A and 14.7% of G, with a slight A + T rich feature (60.5%). Within the control region, we identified the termination-associated sequence domain (TAS), and the conserved sequence block domains.

Mitochondrial genome of the garfish Hyporhamphus quoyi (Beloniformes: Hemiramphidae) and phylogenetic relationships within Beloniformes based on whole mitogenomes.

Mitochondrial DNA (mtDNA) can provide genome-level information (e.g. mitochondrial genome structure, phylogenetic relationships and codon usage) for analyzing molecular phylogeny and evolution of teleostean species. The species in the order Beloniformes have commercial importance in recreational fisheries. In order to further clarify the phylogenetic relationship of these important species, we determined the complete mitochondrial genome (mitogenome) of garfish Hyporhamphus quoyi of Hemiramphidae within Beloniformes. The mitogenome was 16,524 bp long and was typical of other teleosts mitogenomes in size and content. Thirteen PCGs started with the typical ATG codon (with exception of the cytochrome coxidase subunit 1 (cox1) gene with GTG). All tRNA sequences could be folded into expected cloverleaf secondary structures except for tRNASer (AGN) which lost a dihydrouracil (DHU) stem. The control region was 866 bp in length, which contained some conserved sequence blocks (CSBs) common to Beloniformes. The phylogenetic relationship between 26 fish Beloniformes species was analyzed based on the complete nucleotide and amino acid sequences of 13 PCGs by two different inference methods (Maximum Likelihood and Bayesian Inference). Phylogenetic analyses revealed Hemiramphidae as the sister group to Exocoetidae and it is a paraphyletic grouping. Our results may provide useful information on mitogenome evolution of teleostean species.

TEFM Enhances Transcription Elongation by Modifying mtRNAP Pausing Dynamics

Regulation of transcription elongation is one of the key mechanisms employed to control gene expression. The single-subunit mitochondrial RNA polymerase (mtRNAP) transcribes mitochondrial genes, such as those related to ATP synthesis. We investigated how mitochondrial transcription elongation factor (TEFM) enhances mtRNAP transcription elongation using a single-molecule optical-tweezers transcription assay, which follows transcription dynamics in real time and allows the separation of pause-free elongation from transcriptional pauses. We found that TEFM enhances the stall force of mtRNAP. Although TEFM does not change the pause-free elongation rate, it enhances mtRNAP transcription elongation by reducing the frequency of long-lived pauses and shortening their durations. Furthermore, we demonstrate how mtRNAP passes through the conserved sequence block II, which is the key sequence for the switch between DNA replication and transcription in mitochondria. Our findings elucidate how both TEFM and mitochondrial genomic DNA sequences directly control the transcription elongation dynamics of mtRNAP.

Mutational analysis of a Drosophila neuroblast enhancer governing nubbin expression during CNS development

SummaryWhile developmental studies of Drosophila neural stem cell lineages have identified transcription factors (TFs) important to cell identity decisions, currently only an incomplete understanding exists of the cis‐regulatory elements that control the dynamic expression of these TFs. Our previous studies have identified multiple enhancers that regulate the POU‐domain TF paralogs nubbin and pdm‐2 genes. Evolutionary comparative analysis of these enhancers reveals that they each contain multiple conserved sequence blocks (CSBs) that span TF DNA‐binding sites for known regulators of neuroblast (NB) gene expression in addition to novel sequences. This study functionally analyzes the conserved DNA sequence elements within a NB enhancer located within the nubbin gene and highlights a high level of complexity underlying enhancer structure. Mutational analysis has revealed CSBs that are important for enhancer activation and silencing in the developing CNS. We have also observed that adjusting the number and relative positions of the TF binding sites within these CSBs alters enhancer function.

Structural characteristics of the control region of the Beaufortia kweichowensis mitochondrial genome

In present study, we identified the structural characteristics of the Beaufortia kweichowensis mtDNA control region using the next-generation sequencing method. Result showed that the control region could be further divided into three parts, including the extended termination associated sequence domain (ETAS), the central conserved domains (CSB-F, CSB-E, CSB-D) and the conserved sequence block domains (CSB-1, CSB-2, CSB-3), and their conserved sequences were identified.

The kinetoplast DNA of the Australian trypanosome, Trypanosoma copemani, shares features with Trypanosoma cruzi and Trypanosoma lewisi

Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids. It consists of a few dozen maxicircles and several thousand minicircles, all catenated topologically to form a two-dimensional DNA network. Minicircles are heterogeneous in size and sequence among species. They present one or several conserved regions that contain three highly conserved sequence blocks. CSB-1 (10 bp sequence) and CSB-2 (8 bp sequence) present lower interspecies homology, while CSB-3 (12 bp sequence) or the Universal Minicircle Sequence is conserved within most trypanosomatids. The Universal Minicircle Sequence is located at the replication origin of the minicircles, and is the binding site for the UMS binding protein, a protein involved in trypanosomatid survival and virulence. Here, we describe the structure and organisation of the kDNA of Trypanosoma copemani, a parasite that has been shown to infect mammalian cells and has been associated with the drastic decline of the endangered Australian marsupial, the woylie (Bettongia penicillata). Deep genomic sequencing showed that T. copemani presents two classes of minicircles that share sequence identity and organisation in the conserved sequence blocks with those of Trypanosoma cruzi and Trypanosoma lewisi. A 19,257 bp partial region of the maxicircle of T. copemani that contained the entire coding region was obtained. Comparative analysis of the T. copemani entire maxicircle coding region with the coding regions of T. cruzi and T. lewisi showed they share 71.05% and 71.28% identity, respectively. The shared features in the maxicircle/minicircle organisation and sequence between T. copemani and T. cruzi/T. lewisi suggest similarities in their process of kDNA replication, and are of significance in understanding the evolution of Australian trypanosomes.

Hemiptera Mitochondrial Control Region: New Sights into the Structural Organization, Phylogenetic Utility, and Roles of Tandem Repetitions of the Noncoding Segment.

As a major noncoding fragment, the control region (CR) of mtDNA is responsible for the initiation of mitogenome transcription and replication. Several structural features of CR sequences have been reported in many insects. However, comprehensive analyses on the structural organization and phylogenetic utility, as well as the role of tandem replications (TRs) on length variation, high A+T content, and shift of base skew of CR sequences are poorly investigated in hemipteran insects. In this study, we conducted a series of comparative analyses, using 116 samples covering all 11 infraorders of the five currently recognized monophyletic groups in the Hemiptera. Several structural elements (mononucleotide stretches containing conserved sequence blocks (CSBs), TRs, and GA-rich region) were identified in the mitochondrial control region in hemipteran insects, without showing a consistent location. The presence and absence of certain specific structural elements in CR sequences show the various structural organizations of that segment among the five monophyletic groups, which indicates the diversification of the control region’s structural organization in Hemiptera. Among the many groups within Hemiptera, eight monophyletic groups and three consistent phylogenetic trees were recovered, using CSBs datasets by maximum likelihood and Bayesian methods, which suggests the possible utility of CR sequences for phylogenetic reconstruction in certain groups of Hemiptera. Statistical analyses showed that TRs may contribute to the length variation, high AT content, and the shift of base skewing of CR sequences toward high AT content in the Hemiptera. Our findings enrich the knowledge of structural organization, phylogenetic utility, and roles of tandem replication of hemipteran CR, and provide a possible framework for mitochondrial control region analyses in hemimetabolous insects.

Structure and cis-regulatory analysis of a Drosophila grainyhead neuroblast enhancer.

Evolutionary analysis of cis-regulatory DNA reveals that enhancers consist of clusters of conserved sequence blocks (CSBs) that are made up of both unique and repeated sequence elements. This study seeks to address the basis for spatial and temporal regulation of neuroblas. A search for temporally restricted CNS NB enhancers identified one within the transcription factor grainyhead (grh) gene locus. The intronic enhancer, grh-15, contains two separable semi-autonomous activities, one that drives expression predominantly within the developing brain NBs and another in ventral cord NBs. To gain insight into the function of the CSBs constituting the brain-specific enhancer, we have systematically deleted each CSB and compared the activity of the altered enhancer to that of the full brain-specific enhancer. While our results indicate that information regulating enhancer activity is highly redundant, we have found that individual CSBs convey expression in subsets of larval lineages that are generated from either Type I or Type II NBs. These studies also highlight how evolutionary sequence conservation can be used as a guide the functional analysis of cis-regulatory DNA.