The complete sequence of the mitochondrial genome of Daphnia pulex (Cladocera: Crustacea).

The sequence of the mitochondrial DNA (mtDNA) of the branchiopod crustacean Daphnia pulex has been completed. It is 15333bp with an A+T content of 62.3%, and contains the typical complement of 13 protein-coding, 22 transfer RNA (tRNA) and two ribosomal RNA (rRNA) genes. Comparison of this sequence with the sequences of the other eight completely sequenced arthropod mtDNAs showed that gene order and orientation are identical to that of Drosophila but different from Artemia due to the rearrangement of two tRNA genes. Nucleotide composition, codon usage, and amino acid composition are very similar in the crustaceans, but divergent from insects and chelicerates which show a much higher bias towards A+T. However, with few exceptions, the mitochondrial proteins of Daphnia are more similar to those of the dipteran insects (Drosophila and Anopheles) than to those of Artemia, at both the nucleotide and amino acid levels, suggesting that Artemia mtDNA is evolving at an accelerated rate. These results also show that sequence evolution and the evolution of nucleotide composition can be decoupled. Analysis of nucleotide substitution patterns in COII showed that there has been an unbiased acceleration of the overall substitution rate in Artemia. In contrast, the accelerated substitution rate in Apis is due partly to extreme A+T mutation pressure. Secondary structures are proposed for the Daphnia tRNAs and rRNAs. The tRNAs are similar to those of other arthropods but tend to have TPsiC arms that are only 4bp long. The rRNA secondary structures are similar to those proposed for insects except for the absence of a small number of helices in Daphnia. Phylogenetic analysis of second codon positions grouped Daphnia with Artemia, as expected, despite the latter's accelerated divergence rate. In contrast, the unusual pattern of mtDNA divergence in Apis led to a topology in which the holometabolous insects (Anopheles, Drosophila, Apis) appeared to be paraphyletic with respect to the hemimetabolous insect, Locusta, due to the early branching of Apis.     

The sequence,organization, and evolution of the Locusta migratoria mitochondrial genome

The sequencing of the cloned Locusta migratoria mitochondrial genome has been completed. The sequence is 15,722 by in length and contains 75.3% A+T, the lowest value in any of the five insect mitochondrial sequences so far determined. The protein coding genes have a similar A+T content (74.1%) but are distinguished by a high cytosine content at the third codon position. The gene content and organization are the same as in Drosophila yakuba except for a rearrangement of the two tRNA genes tRNA^lys and tRNA^asp. The A+T-rich region has a lower A+T nucleotide content than in other insects, and this is largely due to the presence of two G+C-rich 155-bp repetitive sequences at the 5 end of this section and the beginning of the adjacent small rRNA gene. The sizes of the large and small rRNA genes are 1,314 and 827 bp, respectively, and both sequences can be folded to form secondary structures similar to those previously predicted for Drosophila. The tRNA genes have also been modeled and these show a strong resemblance to the dipteran tRNAs, all anticodons apparently being conserved between the two species. A comparison of the protein coding nucleotide sequences of the locust DNA with the homologous sequences of five other arthropods (Drosophila yakuba, Anopheles quadrimaculatus, Anopheles gambiae, Apis mellifera, and Artemia franciscana) was performed. The amino acid composition of the encoded proteins in Locusta is similar to that of Drosophila, with a Dayhoff distance twice that of the distance between the fruit fly and the mosquitoes. A phylogenetic analysis revealed the locust genes to be more similar to those of the Dipterans than to those of the honeybee at both the nucleotide and amino acid levels. A comparative analysis of tRNA orders, using crustacean mtDNAs as outgroups, supported this. This high level of divergence in the Apis genome has been noted elsewhere and is possibly an effect of directional mutation pressure having resulted in an accelerated pattern of sequence evolution. If the general assumption that the Holometabola are monophyletic holds, then these results emphasize the difficulties of reconstructing phylogenies that include lineages with variable substitution rates and base composition biases. The need to exercise caution in using information about tRNA gene orders in phylogenetic analysis is also illustrated. However, if the honeybee sequence is excluded, the correspondence between the other five arthropod sequences supports the findings of previous studies which have endorsed the use of mtDNA sequences for studies of phylogeny at deep levels of taxonomy when mutation rates are equivalent. Correspondence to: P.K. Flook     

Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster

Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), and tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile)and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region- like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region- like sequences has long persisted in snake mtDNAs, possibly since the original insertion of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T psi C arm of snake mitochondrial tRNAs.      

Analysis of a drosophila tRNA gene cluster: two tRNALeu genes contain intervening sequences

A recombinant DNA phage containing a cluster of Drosophila melanogaster tRNA genes has been isolated and analyzed. The insert of this phage has been mapped by in situ hybridization to chromosomal region 50AB, a known tRNA site. Nucleotide sequencing of the entire Drosophila tRNA coding region reveals seven tRNA genes spanning 2.5 kb of chromosomal DNA. This cluster is separated from other tRNA regions on the chromosome by at least 2.7 kb on one side, and 9.6 kb on the other. Two tRNA genes are nearly identical and contain intervening sequences of length 38 and 45 bases, respectively, in the anticodon loop. These two genes are assigned to be tRNALeu genes because of significant sequence homology with yeast tRNA3Leu, and secondary structure homology with yeast tRNA3Leu intervening sequence. In addition, an 8 base sequence (AAAAUCUU) is conserved in the same location in the intervening sequences of Drosophila tRNALeu genes and a yeast tRNA3Leu gene. Similar sequenes occur in all other tRNAs containing intervening sequences. The remaining five genes are identical tRNAIle genes, which are also identical to a tRNAIle gene from chromosomal region 42A. The 5' flanking regions are only weakly homologous, but each set of isoacceptors contains short regions of strong homology approximately 20 nucleotides preceding the tRNA coding sequences: GCNTTTTG preceding tRNAIle genes; and GANTTTGG preceding tRNALeu genes. The genes are irregularly distributed on both DNA strands; spacing regions are divergent in sequence and length.     

Use of a PCR method for controlling pure-breeding of honeybees Apis mellifera mellifera L. in the southern Urals

A key problem of honeybee (Apis mellifera mellifera) breeding in the Southern Urals is its cross-breeding with the Caucasian honeybee Apis mellifera caucasica. Mitochondrial DNA (mtDNA) in these subspecies differ in the length of a fragment localized between genes CO-I and CO-II, which can be used as a marker. A pair of 20-mer primers for PCR was chosen by means of computer design in order to determine the fragment size in both of the subspecies. The amplified fragment was shown to have a length of 350 bp in A. m. caucasica and 600 bp in A. m. mellifera. The difference in length results from the different ratio between two main elements P and Q, which comprise a major part of this sequence in these subspecies: a copy of P element and two copies of Q element in A. m. mellifera, and a copy of Q element only in A. m. caucasica. This sharply defined distinction allows us to use PCR for differentiating the subspecies, estimating the heterogeneity in the colonies, and rejecting queens in the selection process because of the maternal inheritance of the studied character. The nucleotide sequence of the amplified mtDNA fragment of A. m. mellifera was determined.     

Transfer RNA genes in Drosophila mitochondrial DNA: related 5'' flanking sequences and comparisons to mammalian mitochondrial tRNA genes.

Genes for tRNAgly and tRNAserUCN have been identified within sequences of mtDNA of Drosophila yakuba. The tRNAgly gene lies between the genes for cytochrome c oxidase subunit III and URF3, and all three of these genes are contained in the same strand of the mtDNA molecule. The tRNAserUCN gene is adjacent to the URF1 gene. These genes are contained in opposite strands of the mtDNA molecule and their 3' ends overlap. The structures of the tRNAgly and tRNAserUCN genes, and of the four tRNA genes of D. yakuba mtDNA reported earlier (tRNAile, tRNAgln, tRNAf-met and tRNAval) are compared to each other, to non-organelle tRNAs, and to corresponding mammalian mitochondrial tRNA genes. Within 19 nucleotides upstream from the 5' terminal nucleotide of each of the Drosophila mitochondrial tRNAgly, tRNAserUCN, tRNAile, tRNAgln and tRNAf-met genes occurs the sequence 5'TTTATTAT, or a sequence differing from it by one nucleotide substitution. Upstream from this octanucleotide sequence, and separated from it by 3, 4 and 11 nucleotides, respectively, in the 5' flanking regions of the tRNAile, tRNAserUCN and tRNAgly genes occurs the sequence 5'GATGAG.     

Using iodinated single-stranded M13 probes to facilitate rapid DNA sequence analysis--nucleotide sequence of a mouse lysine tRNA gene.

From a recombinant lambda phage, we have determined a 387 bp sequence containing a mouse lysine tRNA gene. The putative lys tRNA (anticodon UUU) differs from rabbit liver lys tRNA at five positions. The flanking regions of the mouse gene are not generally homologous to published human and Drosophila lys tRNA genes. However, the mouse gene contains a 14 bp region comprising 13 A-T base pairs, 30-44 bp from the 5' end of the coding region. Cognate A-T rich regions are present in human and Drosophila genes. The coding region is flanked by two 11 bp direct repeats, similar to those associated with alu family sequences. The sequence was determined by a "walking" protocol that employs, as a novel feature, iodinated single-stranded M13 probes to identify M13 subclones which contain sequences partially overlapping and contiguous to an initially determined sequence. The probes can also be used to screen lambda phage and in Southern and dot blot experiments.     

The cytochrome b region in the mitochondrial DNA of the ant Tetraponera rufoniger: Sequence divergence in hymenoptera may be associated with nucleotide content

Polymerase chain reaction (PCR) followed by sequencing of single-stranded DNA yielded sequence information from the cytochrome b (cyt b) region in mitochondrial DNA from the ant Tetraponera rufoniger. Compared with the cyt b genes from Apis mellifera, Drosophila melanogaster, and D. yakuba, the overall A + T content (A + T%) of that of T. rufoniger is lower (69.9% vs 80.7%, 74.2%, and 73.9%, respectively) than those of the other three. The codon usage in the cyt b gene of T. rufoniger is biased although not as much as in A. mellifera, D. melanogaster, and D. yakuba; T. rufoniger has eight unused codons whereas D. melanogaster, D. yakuba, and A. mellifera have 21, 20, and 23, respectively. The inferred cyt b polypeptide chain (PPC) of T. rufoniger has diverged at least as much from a common ancestor with D. yakuba as has that of A. mellifera (3.5 vs 2.9). Despite the lower A + T%, the relative frequencies of amino acids in the cyt b PPC of T. rufoniger are significantly (P < 0.05) associated with the content of adenine and thymine (A + T%) and size of codon families. The mitochondrially located cytochrome oxidase subunit 11 genes (CO-II) of endopterygote insects have significantly higher average A + T% (75%) than those of exopterygous (69%o) and paleopterous (69%) insects. The increase in A + T% of endopterygote insects occurred in Upper Carboniferous and coincided with a significant acceleration of PPC divergence. However, acceleration of PPC divergence is not significantly correlated with the increase of the A + T% (P > 0.1). The high A + T%, the biased codon usage, and the increased PPC divergence of Hymenoptera can in that respect most easily be explained by directional mutation pressure which began in the Upper Carboniferous and still occurs in most members of the order. Given the roughly identical A + T% of the cyt b and CO-II genes from the other insects whose DNA sequences are known (A. mellifera, D. melanogaster, and D. yakuba), it seems most likely that the A + T% of T. rufoniger declined secondarily within the last 100 Myr as a result of a reduced directional mutation pressure.Abbreviations Myr million years - mtDNA mitochondrial DNA - scnDNA single-copy nuclear DNA - A adenine - C cytosine - G guanine - T thymine - A + T% content of A and T - PPC polypeptide chain - cyt b cytochrome b - CO-I cytochrome oxidase sub-unit I - CO-II cytochrome oxidase subunit II - ND1 NADH dehydrogenase subunit 1 - ND6 NADH dehydrogenase subunit 6 - tRNA _infUCN ^supSer ucN transfer RNA for serine with a UCN anticodon Correspondence to: L.S. Jermiin     

The Mitochondrial Genome of the Honeybee Apis Mellifera: Complete Sequence and Genome Organization

The complete sequence of honeybee (Apis mellifera) mitochondrial DNA is reported being 16,343 bp long in the strain sequenced. Relative to their positions in the Drosophila map, 11 of the tRNA genes are in altered positions, but the other genes and regions are in the same relative positions. Comparisons of the predicted protein sequences indicate that the honeybee mitochondrial genetic code is the same as that for Drosophila; but the anticodons of two tRNAs differ between these two insects. The base composition shows extreme bias, being 84.9% AT (cf. 78.6% in Drosophila yakuba). In protein-encoding genes, the AT bias is strongest at the third codon positions (which in some cases lack guanines altogether), and least in second codon positions. Multiple stepwise regression analysis of the predicted products of the protein-encoding genes shows a significant association between the numbers of occurrences of amino acids and %T in codon family, but not with the number of codons per codon family or other parameters associated with codon family base composition. Differences in amino acid abundances are apparent between the predicted Apis and Drosophila proteins, with a relative abundance in the Apis proteins of lysine and a relative deficiency of alanine. Drosophila alanine residues are as often replaced by serine as conserved in Apis. The differences in abundances between Drosophila and Apis are associated with %AT in the codon families, and the degree of divergence in amino acid composition between proteins correlates with the divergence in %AT at the second codon positions. Overall, transversions are about twice as abundant as transitions when comparing Drosophila and Apis protein-encoding genes, but this ratio varies between codon positions. Marked excesses of transitions over chance expectation are seen for the third positions of protein-coding genes and for the gene for the small subunit of ribosomal RNA. For the third codon positions the excess of transitions is adequately explained as due to the restriction of observable substitutions to transitions for conserved amino acids with two-codon families; the excess of transitions over expectation for the small ribosomal subunit suggests that the conservation of nucleotide size is favored by selection.     

The complete mitochondrial genome of the Asiatic cavity-nesting honeybee Apis cerana (Hymenoptera: Apidae)

In the present study, we determined the complete mitochondrial DNA (mtDNA) sequence of Apis cerana, the Asiatic cavity-nesting honeybee. We present here an analysis of features of its gene content and genome organization in comparison with Apis mellifera to assess the variation within the genus Apis and among main groups of Hymenoptera. The size of the entire mt genome of A. cerana is 15,895 bp, containing 2 ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes and one control region. These genes are transcribed from both strands and have a nucleotide composition high in A and T. The contents of A+T of the complete genomes are 83.96% for A. cerana. The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. There are a total of 3672 codons in all 13 protein-coding genes, excluding termination codons. The most frequently used amino acid is Leu (15.52%), followed by Ile (12.85%), Phe (10.10%), Ser (9.15%) and Met (8.96%). Intergenic regions in the mt genome of A. cerana are 705 bp in total. The order and orientation of the gene arrangement pattern is identical to that of A. mellifera, except for the position of the tRNA-Ser(AGN) gene. Phylogenetic analyses using concatenated amino acid sequences of 13 protein-coding genes, with three different computational algorithms (NJ, MP and ML), all revealed two distinct groups with high statistical support, indicating that A. cerana and A. mellifera are two separate species, consistent with results of previous morphological and molecular studies. The complete mtDNA sequence of A. cerana provides additional genetic markers for studying population genetics, systematics and phylogeographics of honeybees.     

基于系统发生探讨绿带翠凤蝶和西番翠凤蝶的分类地位(英文)

绿带翠凤蝶和西番翠凤蝶的分类问题存在一定的争议。应用分子系统学方法对这一问题进行了研究。对6个不同地区的24个绿带翠凤蝶、2个地区的16个西番翠凤蝶个体的COI(579bp)和COII(655bp)基因测序,绿带翠凤蝶与西番翠凤蝶的遗传距离为0至0.6%,共获得了15个单倍型。结果显示这些单倍型不能形成各自独立的单系群,因此认为绿带翠凤蝶和西番翠凤蝶为近期分化的两个种。     

Genes for tRNALys5 from Drosophila melanogaster.

The sequences of two cloned genes from Drosophila which hybridize with tRNALys5 are reported. One gene, in plasmid pDt39, has a sequence which corresponds to the sequence of tRNA. The other gene, in pDt59R, differs in three nucleotides pairs. Both plasmids are transcribed in vitro with extracts of Drosophila Kc cells to give full-sized tRNA precursors with four additional nucleotides at the 5'-end as well as truncated molecules containing 35 nucleotides. This premature termination occurs in a block of four T residues within the mature coding region. Sequences flanking the tRNA genes show little in common except for the blocks of five or more T-residues beyond the 3'-end of the gene. pDt39 hybridizes to 84AB on the polytene chromosomes of Drosophila and pDt59R hybridizes to 29A.     

Evoluation of Drosophila mitochondrial DNAs. Analysis of heteroduplex molecules.

We have mapped the single block of non-homologous sequences and measured the extent and distribution of base-pair substitutions within the homologous sequences in Drosophila melanogaster: Drosophila virilis heteroduplex mitochondrial DNAs (mtDNAs). Of the 4.8 kilobases long, unusually (A + T)-rich region in D. melanogaster mtDNA, only 0.5 kilobases can react with related, but not identical sequences in D. virilis mtDNA, while the rest (4.3 kilobases in the long arm of a heteroduplex loop) is replaced by a shorter, non-homologous region (1.0 kilobases in the short arm of the loop). No additional heterologous regions are evident. Homologous sequences have accumulated on the average 15.5% base-pair changes. Regionally, these substitutions are relatively uniformly distributed (14.5--16.5%) except for a single, more conserved region (10--13%), which presumably represents the ribosomal cistrons. The lack of general sequence stability suggests that the invariant topographic organization of the nucleotide sequence, previously recognized among Drosophila mtDNAs, is under more stringent selection than the sequence per se.     

Transfer RNA genes from Dictyostelium discoideum are frequently associated with repetitive elements and contain consensus boxes in their 5' and 3'-flanking regions.

A total of 68 different tRNA genes from the cellular slime mold Dictyostelium discoideum have been isolated and characterized. Although these tRNA genes show features common to typical nuclear tRNA genes from other organisms, several unique characteristics are apparent: (1) the 5'-proximal flanking region is very similar for most of the tRNA genes; (2) more than 80% of the tRNA genes contain an "ex-B motif" within their 3'-flanking region, which strongly resembles characteristics of the consensus sequence of a T-stem/T-loop region (B-box) of a tRNA gene; (3) probably more than 50% of the tRNA genes in certain D. discoideum strains are associated with a retrotransposon, termed DRE (Dictyostelium repetitive element), or with a transposon, termed Tdd-3 (Transposon Dictyostelium discoideum). DRE always occurs 50 (+/- 3) nucleotides upstream and Tdd-3 always occurs 100 (+/- 20) nucleotides downstream from the tRNA gene. D. discoideum tRNA genes are organized in multicopy gene families consisting of 5 to 20 individual genes. Members of a particular gene family are identical within the mature tRNA coding region while flanking sequences are idiosyncratic.     

The complete mitochondrial genome of the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae)

We determined the complete nucleotide sequence of the mitogenome from Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). The 15,566 bp circular genome contains atypical gene organization and order for lepidopteran mitogenomes. The mitogenome contains the lowest A+T content (80.16%) among the known lepidopteran mitogenome sequences. An unusual feature is the occurrence of more Ts than As, with a slightly negative AT skewness (−0.021), in the composition of the major genome strand. All protein-coding genes are initiated by ATN codons, except for cytochrome oxidase subunit I, which is proposed by the TTAG sequence as observed in other lepidopterans. All transfer RNAs (tRNAs) have a typical clover-leaf structure of mitochondrial tRNA, except for tRNA ^Ser (AGN) , the DHU arm of which could not form a stable stem-loop structure. Two aligned sequence blocks with a length of more than 50 bp and 90% of the sequence identity were identified in the A+T-rich region of the Saturniidae and Bombycoidae species.     

Complete mitochondrial genome of the nerippe fritillary butterfly, Argynnis nerippe (Lepidoptera: Nymphalidae)

The complete mitochondrial genome sequence of the nerippe fritillary butterfly, Argynnis nerippe, which is listed as an endangered species in Korea, is described with an emphasis on the A+T-rich region. The 15,140-bp long circular molecule consisted of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and 1 control region, known in insect as the A+T-rich region, as found in typical metazoans. The 329-bp long A+T-rich region located between srRNA and tRNA(Met) possessed the highest A/T content (95.7%) than any other region of the genome. Along with the several conserved sequences found typically in the lepidopteran insects the genome contained one tRNA(Met)-like and tRNA(Leu)(UUR)-like sequence in the A+T-rich region.     

Extensive microheterogeneity of serine tRNA genes from Drosophila melanogaster

The nucleotide sequences of nine genes corresponding to tRNA(Ser)4 or tRNA(Ser)7 of Drosophila melanogaster were determined. Eight of the genes compose the major tRNA(Ser)4,7 cluster at 12DE on the X chromosome, while the other is from 23E on the left arm of chromosome 2. Among the eight X-linked genes, five different, interrelated, classes of sequence were found. Four of the eight genes correspond to tRNA(Ser)4 and tRNA(Ser)7 (which are 96% homologous), two appear to result from single crossovers between tRNA(Ser)4 and tRNA(Ser)7 genes, one is an apparent double crossover product, and the last differs from a tRNA(Ser)4 gene by a single C to T transition at position 50. The single autosomal gene corresponds to tRNA(Ser)7. Comparison of a pair of genes corresponding to tRNA(Ser)4 from D. melanogaster and Drosophila simulans showed that, while gene flanking sequences may diverge considerably by accumulation of point changes, gene sequences are maintained intact. Our data indicate that recombination occurs between non-allelic tRNA(Ser) genes, and suggest that at least some recombinational events may be intergenic conversions.