Extrachromosomal rDNA of Tetrahymena thermophila is not a perfect palindrome

We have determined the restriction-endonuclease-cleavage map and the nucleotide sequence of the central 1.4 kb fragment of the macronuclear extrachromosomal rDNA of Tetrahymena thermophila. These data demonstrate that this molecule is not a perfect palindrome, having a 29 bp AT-rich non-palindromic sequence at its center. This observation is important in determining the mechanism by which a single chromosomally integrated rRNA gene in the micronucleus is rearranged and amplified during sexual development to yield multiple copies of extrachromosomal rDNA in the macronucleus.     

Isolation and characterization of Chinese hamster ribosomal DNA clones

We have examined the ribosomal RNA (rRNA) genes of the Chinese hamster ovary (CHO) cell line. A partial EcoRI library of genomic CHO DNA was prepared using lambda Charon-4A. We isolated two recombinants containing the region transcribed as 45S pre-rRNA and 13 kb of external spacer flanking 5' and 3' to the transcribed region. These sequences show restriction site homology with the vast majority of the genomic sequences complementary to rRNA. In addition to this form of rDNA, Southern blot analysis of EcoRI-cut CHO genomic DNA reveals numerous minor fragments ranging from 2 to 19 kb which are complementary to 18S rRNA. We isolated one clone which contains the 18S rRNA gene and sequences 5' which appear to contain length heterogeneity within the non-transcribed spacer region. We have nine additional cloned EcoRI fragments in which the homology with 18S rRNA is limited to a 0.9-kb EcoRI-HindIII fragment. This EcoRI-HindIII fragment is present in each of the cloned EcoRI fragments, and is flanked on both sides by apparently nonribosomal sequences which bear little restriction site homology with each other or the major cloned rDNA repeat.     

Insertion of a long KpnI family member within a mitochondrial-DNA-like sequence present in the human nuclear genome

Structural analysis of a phage lambda Charon 4A clone carrying one of the human nuclear mitochondrial(mut)-DNA-like sequences revealed that a KpnI-family member (KpnI 5.5-kb DNA) is inserted within this sequence. The inserted KpnI 5.5-kb DNA contains several possible polyadenylation signal sequences followed by an A-rich sequence at its 3' end and is flanked by perfect 13-bp direct repeats of the duplicated mtDNA-like sequences. These structures strongly suggest that the KpnI 5.5-kb DNA is a mobile element. Comparison of the 5' terminal sequences of the KpnI 5.5-kb DNA and four other long KpnI-family DNAs so far examined, using the predicted general promoter sequence for eukaryotic tRNAs, indicates that they contain the consensus sequences for the split internal RNA polymerase III control region.     

Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands

We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.     

Probing recalcitrant problems in polyclad evolution and systematics with novel mitochondrial genome resources

For their apparent morphological simplicity, the Platyhelminthes or “flatworms” are a diverse clade found in a broad range of habitats. Their body plans have however made them difficult to robustly classify. Molecular evidence is only beginning to uncover the true evolutionary history of this clade. Here we present nine novel mitochondrial genomes from the still undersampled orders Polycladida and Rhabdocoela, assembled from short Illumina reads. In particular we present for the first time in the literature the mitochondrial sequence of a Rhabdocoel, Bothromesostoma personatum (Typhloplanidae, Mesostominae). The novel mitochondrial genomes examined generally contained the 36 genes expected in the Platyhelminthes, with all possessing 12 of the 13 protein-coding genes normally found in metazoan mitochondrial genomes (ATP8 being absent from all Platyhelminth mtDNA sequenced to date), along with two ribosomal RNA genes. The majority presented possess 22 transfer RNA genes, and a single tRNA gene was absent from two of the nine assembled genomes. By comparison of mitochondrial gene order and phylogenetic analysis of the protein coding and ribosomal RNA genes contained within these sequences with those of previously sequenced species we are able to gain a firm molecular phylogeny for the inter-relationships within this clade.Our phylogenetic reconstructions, using both nucleotide and amino acid sequences under several models and both Bayesian and Maximum Likelihood methods, strongly support the monophyly of Polycladida, and the monophyly of Acotylea and Cotylea within that clade. They also allow us to speculate on the early emergence of Macrostomida, the monophyly of a “Turbellarian-like” clade, the placement of Rhabditophora, and that of Platyhelminthes relative to the Lophotrochozoa (=Spiralia). The data presented here therefore represent a significant advance in our understanding of platyhelminth phylogeny, and will form the basis of a range of future research in the still-disputed classifications within this taxon.     

Complete mitochondrial genome sequences of three bats species and whole genome mitochondrial analyses reveal patterns of codon bias and lend support to a basal split in Chiroptera

Order Chiroptera is a unique group of mammals whose members have attained self-powered flight as their main mode of locomotion. Much speculation persists regarding bat evolution; however, lack of sufficient molecular data hampers evolutionary and conservation studies. Of ~ 1200 species, complete mitochondrial genome sequences are available for only eleven. Additional sequences should be generated if we are to resolve many questions concerning these fascinating mammals. Herein, we describe the complete mitochondrial genomes of three bats: Corynorhinus rafinesquii, Lasiurus borealis and Artibeus lituratus. We also compare the currently available mitochondrial genomes and analyze codon usage in Chiroptera. C. rafinesquii, L. borealis and A. lituratus mitochondrial genomes are 16438 bp, 17048 bp and 16709 bp, respectively. Genome organization and gene arrangements are similar to other bats. Phylogenetic analyses using complete mitochondrial genome sequences support previously established phylogenetic relationships and suggest utility in future studies focusing on the evolutionary aspects of these species. Comprehensive analyses of available bat mitochondrial genomes reveal distinct nucleotide patterns and synonymous codon preferences corresponding to different chiropteran families. These patterns suggest that mutational and selection forces are acting to different extents within Chiroptera and shape their mitochondrial genomes.     

Complete nucleotide sequence and gene organization of the mitochondrial genome of Paa spinosa (Anura: Ranoidae)

The mt genome of Paa spinosa (Anura: Ranoidae) is a circular molecule of 18,012 bp in length, containing 38 genes (including an extra copy of tRNA-Met gene). This mt genome is characterized by three distinctive features: a cluster of rearranged tRNA genes (LTPF tRNA gene cluster), a tandem duplication of tRNA-Met gene (Met1 and Met2), and distinct repeat regions at both 5′ and 3′-sides in the control region. Comparing the locations and the sequences of all tRNA-Met genes among Ranoidae, and constructing NJ tree of the nucleotide of those tRNA-Met genes, we suggested a tandem duplication of tRNA-Met gene can be regarded as a synapomorphy of Dicroglossinae. To further investigate the phylogenetic relationships of anurans, phylogenetic analyses (BI, ML and MP) based on the nucleotide dataset and the corresponding amino acid dataset of 11 protein-coding genes (except ND5 and ATP8) arrived at the similar topology.     

Complete mitochondrial genome sequence of bighead croaker Collichthys niveatus (Perciformes, Sciaenidae): a mitogenomic perspective on the phylogenetic relationships of Pseudosciaeniae

The monophyly and phylogenetic relationships of Pseudosciaeniae have long been controversial. Here we describe the mitochondrial genome (mitogenome) sequence of Collichthys niveatus. It is a circular double-stranded DNA molecule of 16,450 base pairs (bp) in length with a standard set of 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), 13 protein-coding genes as well as a non-coding control region. The mitogenome of C. niveatus shared common features with those of other bony fishes in terms of gene arrangement, base composition, and tRNA structures. The C. niveatus mitogenome exhibited pronounced strand-specific asymmetry in nucleotide composition, which was also reflected in the codon usage of genes oriented in opposite directions. Contrary to the typical structure of the control region, the central conserved blocks (CSB-D, -E, and -F) could not be detected in C. niveatus mitogenome. Phylogenetic analysis based on whole mitogenome sequences provided strong support for the monophyly of Pseudosciaeniae, and sister-group relationships of C. niveatus + Collichthys lucidus and Larimichthys crocea + Larimichthys polyactis, which was consistent with the traditional taxonomy. Unexpected divergence was found in two C. niveatus mitogenomes and several hypotheses were proposed to explain this observation including misidentification and introgressive hybridization between C. niveatus and L. polyactis, and polyphyletic origin of C. niveatus. We considered species misidentification to be the main hypothesis. However, additional data is essential to test these proposed hypotheses.     

The complete mitochondrial genome of Sasakia funebris (Leech) (Lepidoptera: Nymphalidae) and comparison with other Apaturinae insects

Sasakia funebris, a member of the lepidopteran family, Nymphalidae (superfamily Papilionoidea) is a rare species and is found only in some areas of South China. In this study, the 15,233 bp long complete mitochondrial genome of S. funebris was determined, and harbors the gene arrangement identical to all other sequenced lepidopteran insects. The nucleotide composition of the genome is highly A + T biased, accounting for 81.2%. All protein-coding genes (PCGs) start with typical ATN codons, except for COI which begins with the CGA codon. All tRNAs have a typical clover-leaf secondary structure, except for tRNA^Ser(AGN), the dihydrouridine (DHU) arm of which forms a simple loop. The S. funebris A + T-rich region of 370 bp contains several features common to the Lepidoptera insects, including the motif ATAGA followed by a 19 bp poly-T stretch, and two tandem repeats consisting of 18 bp repeat units and 14 bp repeat units. The phylogenetic analyses of Apaturinae based on mitogenome sequences showed: (S. funebris + Sasakia charonda) + (Apatura metis + Apatura ilia). This result is consistent with the morphological classification.     

The complete mitochondrial genome of Microtus fortis calamorum (Arvicolinae, Rodentia) and its phylogenetic analysis

Microtus fortis is a special resource of rodent in China. It is a promising experimental animal model for the study on the mechanism of Schistosome japonicum resistance. The first complete mitochondrial genome sequence for Microtus fortis calamorum, a subspecies of M. fortis (Arvicolinae, Rodentia), was reported in this study. The mitochondrial genome sequence of M. f. calamorum (Genbank: JF261175) showed a typical vertebrate pattern with 13 protein coding genes, 2 ribosomal RNAs, 22 transfer RNAs and one major noncoding region (CR region).The extended termination associated sequences (ETAS-1 and ETAS-2) and conserved sequence block 1 (CSB-1) were found in the CR region. The putative origin of replication for the light strand (O(L)) of M. f. calamorum was 35bp long and showed high conservation in stem and adjacent sequences, but the difference existed in the loop region among three species of genus Microtus. In order to investigate the phylogenetic position of M. f. calamorum, the phylogenetic trees (Maximum likelihood and Bayesian methods) were constructed based on 12 protein-coding genes (except for ND6 gene) on H strand from 16 rodent species. M. f. calamorum was classified into genus Microtus, Arvcicolinae for the highly phylogenetic relationship with Microtus kikuchii (Taiwan vole). Further phylogenetic analysis results based on the cytochrome b gene ranged from M. f. calamorum to one of the subspecies of M. fortis, which formed a sister group of Microtus middendorfii in the genus Microtus.     

The complete sequence of the mitochondrial genome of the African Penguin (Spheniscus demersus)

The complete mitochondrial genome of the African Penguin (Spheniscus demersus) was sequenced. The molecule was sequenced via next generation sequencing and primer walking. The size of the genome is 17,346 bp in length. Comparison with the mitochondrial DNA of two other penguin genomes that have so far been reported was conducted namely; Little blue penguin (Eudyptula minor) and the Rockhopper penguin (Eudyptes chrysocome). This analysis made it possible to identify common penguin mitochondrial DNA characteristics. The S. demersus mtDNA genome is very similar, both in composition and length to both the E. chrysocome and E. minor genomes. The gene content of the African penguin mitochondrial genome is typical of vertebrates and all three penguin species have the standard gene order originally identified in the chicken. The control region for S. demersus is located between tRNA-Glu and tRNA-Phe and all three species of penguins contain two sets of similar repeats with varying copy numbers towards the 3′ end of the control region, accounting for the size variance. This is the first report of the complete nucleotide sequence for the mitochondrial genome of the African penguin, S. demersus. These results can be subsequently used to provide information for penguin phylogenetic studies and insights into the evolution of genomes.     

The complete mitochondrial genome of Temminck's ground pangolin (Smutsia temminckii; Smuts, 1832) and phylogenetic position of the Pholidota (Weber, 1904)

Temmincki's ground pangolin is primarily a nocturnal mammal belonging to the order Pholidota. The body is covered in hard overlapping scales and these animals find refuge in burrows, feeding only on termites and ants. In this study, the whole mtDNA of Temmincki's ground pangolin was sequenced and the phylogenetic position of Pholidota determined within Eutheria, using whole mtDNA sequences from various representative species. The results indicate that the whole mtDNA of Temmincki's ground pangolin is 16,559 bp long and shared some similarities with the whole mtDNA of the back-bellied tree pangolin and the Chinese pangolin. Phylogenetic analysis indicate that the order Pholidota is closely related and share a recent common ancestor with the order Carnivora rather than with the ant/insect eating order Xenarthra and the group Afrotheria. A time measured phylogeny of Pholidota estimated a split from Carnivora at around 87 mya, followed by a split of the African pangolins from their Asian counterparts such as the Chinese pangolin at around 47 mya. This suggests a Laurasian origin and convergent evolution of the Pholidota with respect to Xenarthra and Afrotheria.     

The phylogeny of Ephemeroptera in Pterygota revealed by the mitochondrial genome of Siphluriscus chinensis (Hexapoda: Insecta)

The mayfly species Siphluriscus chinensis (Siphluriscidae) has valuable structures useful for phylogeny reconstruction, given its putative basal position within the Ephemeroptera. Here its nearly complete mitochondrial genome is sequenced. We built phylogenetic trees through multiple analytical strategies with some other insect mitogenomes. Structurally, the obtained mitochondrial genome of S. chinensis is 16,616 bp in length, ¹ containing 37 genes and an extra trnK-like (trnK2 (AAA)) gene. The 12 PCGs start with typical ATN codons, except the nad1 gene which starts with an unnormalized TTG. Like other known mayfly mitogenomes, the strand bias has negative AT-skew and negative GC-skew. Phylogenetically, our topologies suggest that Odonata is the basally diverged clade in Pterygota; Ephemeroptera is the sister group of the Neoptera; and S. chinensis is indeed the most basal mayfly branch.     

Evolutionary relatedness of mackerels of the genus Scomber based on complete mitochondrial genomes: Strong support to the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as distinct species

Mackerels of the genus Scomber are commercially important species, but their taxonomic status is still controversial. Although previous phylogenetic data support the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as separate species, it is only based on the analysis of partial mitochondrial and nuclear DNA sequences. In an attempt to shed light on this relevant issue, we have determined the complete mitochondrial DNA sequence of S. colias, S. japonicus, and Scomber australasicus. The total length of the mitogenomes was 16,568 bp for S. colias and 16,570 bp for both S. japonicus and S. australasicus. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs, and 22 tRNAs) and organization similar to that observed in Scomber scombrus and most other vertebrates. The major noncoding region (control region) ranged between 865 and 866 bp in length and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Scomber species with regard to other scombrid fish. The major finding of this study is that S. colias and S. japonicus were significantly grouped in distinct lineages within Scomber cluster, which phylogenetically constitutes evidence that they may be considered as separate species. Additionally, molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.     

Complete mitochondrial genomes of five skippers (Lepidoptera: Hesperiidae) and phylogenetic reconstruction of Lepidoptera

We sequenced mitogenomes of five skippers (family Hesperiidae, Lepidoptera) to obtain further insight into the characteristics of butterfly mitogenomes and performed phylogenetic reconstruction using all available gene sequences (PCGs, rRNAs, and tRNAs) from 85 species (20 families in eight superfamilies). The general genomic features found in the butterflies also were found in the five skippers: a high A + T composition (79.3%–80.9%), dominant usage of TAA stop codon, similar skewness pattern in both strands, consistently length intergenic spacer sequence between tRNA^Gln and ND2 (64–87 bp), conserved ATACTAA motif between tRNA^{Ser (UCN)} and ND1, and characteristic features of the A + T-rich region (the ATAGA motif, varying length of poly-T stretch, and poly-A stretch). The start codon for COI was CGA in four skippers as typical, but Lobocla bifasciatus evidently possessed canonical ATG as start codon. All species had the ancestral arrangement tRNA^Asn/tRNA^{Ser (AGN)}, instead of the rearrangement tRNA^{Ser (AGN)}/tRNA^Asn, found in another skipper species (Erynnis). Phylogenetic analyses using all available genes (PCGs, rRNAS, and tRNAs) yielded the consensus superfamilial relationships ((((((Bombycoidea + Noctuoidea + Geometroidea) + Pyraloidea) + Papilionoidea) + Tortricoidea) + Yponomeutoidea) + Hepialoidea), confirming the validity of Macroheterocera (Bombycoidea, Noctuoidea, and Geometroidea in this study) and its sister relationship to Pyraloidea. Within Rhopalocera (butterflies and skippers) the familial relationships (Papilionidae + (Hesperiidae + (Pieridae + ((Lycaenidae + Riodinidae) + Nymphalidae)))) were strongly supported in all analyses (0.98–1 by BI and 96–100 by ML methods), rendering invalid the superfamily status for Hesperioidea. On the other hand, current mitogenome-based phylogeny did not find consistent superfamilial relationships among Noctuoidea, Geometroidea, and Bombycoidea and the familial relationships within Bombycoidea between analyses, requiring further taxon sampling in future studies.