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.     

Drosophila mitochondrial DNA: a novel gene order

Part of the replication origin-containing A+T-rich region of the Drosophila yakuba mtDNA molecule and segments on either side of this region have been sequenced, and the genes within them identified. The data confirm that the small and large rRNA genes lie in tandem adjacent to that side of the A+T-rich region which is replicated first, and establish that a tRNAval gene lies between the two rRNA genes and that URF1 follows the large rRNA gene. The data further establish that the genes for tRNAile, tRNAgln, tRNAf-met and URF2 lie in the order given, on the opposite side of the A+T-rich region to the rRNA genes and, except for tRNAgln, are contained in the opposite strand to the rRNA, tRNAval and URF1 genes. This is in contrast to mammalian mtDNAs where all of these genes are located on the side of the replication origin which is replicated last, within the order tRNAphe, small (12S) rRNA, tRNAval, large (16S) rRNA, tRNAleu, URF1, tRNAile, tRNAgln, tRNAf-met and URF2, and, except tRNAgln, are all contained in the same (H) strand. In D. yakuba URF1 and URF2, the triplet AGA appears to specify an amino acid, which is again different from the situation found in mammalian mtDNAs, where AGA is used only as a rare termination codon.     

Nucleotide sequence of a segment of Drosophila mitochondrial DNA that contains the genes for cytochrome c oxidase subunits II and III and ATPase subunit 6.

The nucleotide sequence of a segment of the mtDNA molecule of Drosophila yakuba has been determined, within which have been identified the genes for tRNAleuUUR, cytochrome c oxidase subunit II (COII), tRNAlys, tRNAasp, URFA6L, ATPase subunit 6 (ATPase6), cytochrome c oxidase subunit III (COIII) and tRNAgly. The genes are arranged in the order given and all are transcribed from the same strand of the molecule in a direction opposite to that in which replication proceeds around the molecule. The tRNAlys gene is unusual among mitochondrial tRNAlys genes in that it contains a CTT anticodon. The triplet AGA is used to specify an amino acid in all of the COII, COIII, ATPase6, and URFA6L genes. However, the AGA codons found in these four polypeptide genes correspond in position to codons which specify nine different amino acids, but never arginine, in the equivalent polypeptide gene which have been sequenced from mtDNAs of mouse, yeast and Zea mays.     

The ribosomal RNA genes of Drosophila mitochondrial DNA.

The nucleotide sequence of a segment of the mtDNA molecule of Drosophila yakuba which contains the A+T-rich region and the small and large rRNA genes separated by the tRNAval gene has been determined. The 5' end of the small rRNA gene was located by S1 protection analysis. In contrast to mammalian mtDNA, a tRNA gene was not found at the 5' end of the D. yakuba small rRNA gene. The small and large rRNA genes are 20.7% and 16.7% G+C and contain only 789 and 1326 nucleotides. The 5' regions of the small rRNA gene (371 nucleotides) and of the large rRNA gene (643 nucleotides) are extremely low in G+C (14.6% and 9.5%, respectively) and convincing sequence homologies between these regions and the corresponding regions of mouse mt-rRNA genes were found only for a few short segments. Nevertheless, the entire lengths of both of the D. yakuba mt-rRNA genes can be folded into secondary structures which are remarkably similar to secondary structures proposed for the rRNAs of mouse mtDNA. The replication origin-containing, A+T-rich region (1077 nucleotides; 92.8% A+T), which lies between the tRNAile gene and the small rRNA gene, lacks open reading frames greater than 123 nucleotides.     

Sequence Evolution of Drosophila Mitochondrial DNA

We have compared nucleotide sequences of corresponding segments of the mitochondrial DNA (mtDNA) molecules of Drosophila yakuba and Drosophila melanogaster, which contain the genes for six proteins and seven tRNAs. The overall frequency of substitution between the nucleotide sequences of these protein genes is 7.2%. As was found for mtDNAs from closely related mammals, most substitutions (86%) in Drosophila mitochondrial protein genes do not result in an amino acid replacement. However, the frequencies of transitions and transversions are approximately equal in Drosophila mtDNAs, which is in contrast to the vast excess of transitions over transversions in mammalian mtDNAs. In Drosophila mtDNAs the frequency of C----T substitutions per codon in the third position is 2.5 times greater among codons of two-codon families than among codons of four-codon families; this is contrary to the hypothesis that third position silent substitutions are neutral in regard to selection. In the third position of codons of four-codon families transversions are 4.6 times more frequent than transitions and A----T substitutions account for 86% of all transversions. Ninety-four percent of all codons in the Drosophila mtDNA segments analyzed end in A or T. However, as this alone cannot account for the observed high frequency of A----T substitutions there must be either a disproportionately high rate of A----T mutation in Drosophila mtDNA or selection bias for the products of A----T mutation. --Consideration of the frequencies of interchange of AGA and AGT codons in the corresponding D. yakuba and D. melanogaster mitochondrial protein genes provides strong support for the view that AGA specifies serine in the Drosophila mitochondrial genetic code.     

Drosophila Melanogaster Mitochondrial DNA: Gene Organization and Evolutionary Considerations

The sequence of a 8351-nucleotide mitochondrial DNA (mtDNA) fragment has been obtained extending the knowledge of the Drosophila melanogaster mitochondrial genome to 90% of its coding region. The sequence encodes seven polypeptides, 12 tRNAs and the 3' end of the 16S rRNA and CO III genes. The gene organization is strictly conserved with respect to the Drosophila yakuba mitochondrial genome, and different from that found in mammals and Xenopus. The high A + T content of D. melanogaster mitochondrial DNA is reflected in a reiterative codon usage, with more than 90% of the codons ending in T or A, G + C rich codons being practically absent. The average level of homology between the D. melanogaster and D. yakuba sequences is very high (roughly 94%), although insertion and deletions have been detected in protein, tRNA and large ribosomal genes. The analysis of nucleotide changes reveals a similar frequency for transitions and transversions, and reflects a strong bias against G + C on both strands. The predominant type of transition is strand specific.     

Analysis of nucleotide substitutions of mitochondrial DNAs in Drosophila melanogaster and its sibling species

To study the rate and pattern of nucleotide substitution in mitochondrial DNA (mtDNA), we cloned and sequenced a 975-bp segment of mtDNA from Drosophila melanogaster, D. simulans, and D. mauritiana containing the genes for three transfer RNAs and parts of two protein- coding genes, ND2 and COI. Statistical analysis of synonymous substitutions revealed a predominance of transitions over transversions among the three species, a finding differing from previous results obtained from a comparison of D. melanogaster and D. yakuba. The number of transitions observed was nearly the same for each species comparison, including D. yakuba, despite the differences in divergence times. However, transversions seemed to increase steadily with increasing divergence time. By contrast, nonsynonymous substitutions in the ND2 gene showed a predominance of transversions over transitions. Most transversions were between A and T and seemed to be due to some kind of mutational bias to which the A + T-rich mtDNA of Drosophila species may be subject. The overall rate of nucleotide substitution in Drosophila mtDNA appears to be slightly faster (approximately 1.4 times) than that of the Adh gene. This contrasts with the result obtained for mammals, in which the mtDNA evolves approximately 10 times faster than single-copy nuclear DNA. We have also shown that the start codon of the COI gene is GTGA in D. simulans and GTAA in D. mauritiana. These codons are different from that of D. melanogaster (ATAA).      

Nucleotide Sequence and Gene Organization of the Starfish Asterina Pectinifera Mitochondrial Genome

The 16,260-bp mitochondrial DNA (mtDNA) from the starfish Asterina pectinifera has been sequenced. The genes for 13 proteins, two rRNAs and 22 tRNAs are organized in an extremely economical fashion, similar to those of other animal mtDNAs, with some of the genes overlapping each other. The gene organization is the same as that for another echinoderm, sea urchin, except for the inversion of a 4.6-kb segment that contains genes for two proteins, 13 tRNAs and the 16S rRNA. Judging from the organization of the protein coding genes, mammalian mtDNAs resemble the sea urchin mtDNA more than that of the starfish. The region around the 3' end of the 12S rRNA gene of the starfish shows a high similarity with those for vertebrates. This region encodes a possible stem and loop structure; similar potential structures occur in this region of vertebrate mtDNAs and also in nonmitochondrial small subunit rRNA. A similar stem and loop structure is also found at the 3' end of the 16S rRNA genes in A. pectinifera, in another starfish Pisaster ochraceus, in vertebrates and in Drosophila, but not in sea urchins. The full sequence data confirm the presumption that AGA/AGG, AUA and AAA codons, respectively, code for serine, isoleucine, and asparagine in the starfish mitochondria, and that AGA/AGG codons are read by tRNA(GCU)(Ser), which possesses a truncated dihydrouridine arm, that was previously suggested from a partial mtDNA sequence. The structural characteristics of tRNAs and possible mechanisms for the change in the mitochondrial genetic code are also discussed.     

Platyhelminth mitochondrial DNA: Evidence for early evolutionary origin of a tRNAserAGN that contains a dihydrouridine arm replacement loop,and of serine-specifying AGA and AGG codons

Summary The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the liver flukeFasciola hepatica (phylum Platyhelminthes, class Trematoda) has been determined, within which have been identified the genes for tRNA^ala, tRNA^asp, respiratory chain NADH dehydrogenase subunit I (ND1), tRNA^asn, tRNA^pro, tRNA^ile, tRNA^lys, ND3, tRNA^serAGN, tRNA^trp, and cytochromec oxidase subunit I (COI). The 11 genes are arranged in the order given and are all transcribed from the same strand of the molecule. The overall order of theF. hepatica mitochondrial genes differs from what is found in other metazoan mtDNAs. All of the sequenced tRNA genes except the one for tRNA^serAGN can be folded into a secondary structure with four arms resembling most other metazoan mitochondrial tRNAs, rather than the tRNAs that contain a TψC arm replacement loop, found in nematode mtDNAs. TheF. hepatica mitochondrial tRNA^serAGN gene contains a dihydrouridine arm replacement loop, as is the case in all other metazoan mtDNAs examined to date. AGA and AGG are found in theF. hepatica mitochondrial protein genes and both codons appear to specify serine. These findings concerningF. hepatica mtDNA indicate that both a dihydrouridine arm replacement loop-containing tRNA^serAGN gene and the use of AGA and AGG codons to specify serine must first have occurred very early in, or before, the evolution of metazoa.     

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.     

Unusual genetic codes and a novel gene structure for tRNA(AGYSer) in starfish mitochondrial DNA

The nucleotide sequence of a 3849-bp fragment of starfish mitochondrial genome was determined. The genes for NADH dehydrogenase subunits 3, 4, 5, and COIII, and three kinds of (tRNA(UCNSer), tRNA(His), and tRNA(AGYSer) were identified by comparing with the genes of other animal mitochondria so far elucidated. The gene arrangement of starfish mitochondrial genome was different from those of vertebrate and insect mitochondrial genomes. Comparison of the protein-encoding nucleotide sequences of starfish mitochondria with those of other animal mitochondria suggested a unique genetic code in starfish mitochondrial genome; both AGA and AGG (arginine in the universal code) code for serine, AUA (isoleucine in the universal code but methionine in most mitochondrial systems) for isoleucine, and AAA (lysine) for asparagine. It was also inferred that these AGA and AGG codons are decoded by serine tRNA(AGYSer) originally corresponding to AGC and AGU codons. This situation is similar to the case of Drosophila mitochondrial genome. Variations in the use of AGA and AGG codons were discussed on the basis of the evolution of animals and decoding capacity of various tRNA(AGYSer) species possessing different sizes of the dihydrouridine (D) arm.     

Nucleotide sequence of Aspergillus nidulans mitochondrial genes coding for ATPase subunit 6, cytochrome oxidase subunit 3, seven unidentified proteins, four tRNAs and L-rRNA.

The complete nucleotide sequence of a 14 kb segment of A. nidulans mtDNA reveals a rather compact organization of genes transcribed from the same strand and coding for two functionally known proteins, seven unidentified polypeptides (URFs), 24 tRNAs and two rRNAs. One of the URFs is located in the intron of the L-rRNA gene and codes for a basic protein of 410 residues. The other URFs are in spacer regions and code for hydrophobic proteins. URFa is homologous to human URF4, and URFb produces a polypeptide of 48 residues resembling the human URF6L product (hydrophobic N-terminus, basic C-terminus). The ATPase subunit 6 genes from mitochondria and E. coli appear to share a common ancestor. The codon frequencies of identified genes and URFs are similar, and codons ending with G or C are rarely used. The structures of tRNAs specific for arginine, asparagine, tyrosine and histidine are deduced from gene sequences.     

鲤鱼线粒体tRNA~(phe)基因结构的特异性

鲤鱼线粒体tRNA~(phe)基因的核酸序列已被测定。在鲸、人、爪蟾、牛、小鼠、鸡和鲤鱼中对此基因序列比较发现在D茎存在一个奇怪的保守结构,然而D茎在其余种类的已经测定的脊椎动物线粒体tRNA基因和细胞质tRNA基因中是极不保守的。这一保守结构包含有13bp碱基,我们将此保守区前7个碱基与真核生物RNA PolⅢ识别的A区相比较,发现在此不同物种的两种序列存在部分的同源性。考虑到tRNA~(phe)基因在线粒体基因组上位于置换环区和线粒体rRNA基因编码区之间这一特殊区域内,我们推测这一奇怪的保守结构可能存在其它更为有意义的功能。     

Complete DNA Sequence of the Mitochondrial Genome of the Black Chiton, Katharina Tunicata

The DNA sequence of the 15,532-base pair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina tunicata has been determined. The 37 genes typical of metazoan mtDNA are present: 13 for protein subunits involved in oxidative phosphorylation, 2 for rRNAs and 22 for tRNAs. The gene arrangement resembles those of arthropods much more than that of another mollusc, the bivalve Mytilus edulis. Most genes abut directly or overlap, and abbreviated stop codons are inferred for four genes. Four junctions between adjacent pairs of protein genes lack intervening tRNA genes; however, at each of these junctions there is a sequence immediately adjacent to the start codon of the downstream gene that is capable of forming a stem-and-loop structure. Analysis of the tRNA gene sequences suggests that the D arm is unpaired in tRNA(ser(AGN)), which is typical of metazoan mtDNAs, and also in tRNA(ser(UCN)), a condition found previously only in nematode mtDNAs. There are two additional sequences in Katharina mtDNA that can be folded into structures resembling tRNAs; whether these are functional genes is unknown. All possible codons except the stop codons TAA and TAG are used in the protein-encoding genes, and Katharina mtDNA appears to use the same variation of the mitochondrial genetic code that is used in Drosophila and Mytilus. Translation initiates at the codons ATG, ATA and GTG. A + T richness appears to have affected codon usage patterns and, perhaps, the amino acid composition of the encoded proteins. A 142-bp non-coding region between tRNA(glu) and CO3 contains a 72-bp tract of alternating A and T.     

The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochrondrial maxi-circle DNA.

A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes.     

The initiator tRNA genes of Drosophila melanogaster: evidence for a tRNA pseudogene

We have isolated four segments of Drosophila melanogaster DNA that hybridize to homologous initiator tRNAMet. Three of the cloned fragments contain initiator tRNA genes, each of which can be transcribed in vitro. The fourth clone, pPW568, contains an initiator tRNA pseudogene which is not transcribed in vitro by RNA polymerase III. The pseudogene is contained in a 1.15 kb DNA fragment. This fragment has the characteristics of dispersed repetitive DNA and hybridizes in situ to at least 30 sites in the Drosophila genome. The arrangement of the initiator tRNA genes we have isolated, is different to that of other Drosophila tRNA gene families. The initiator tRNA genes are not clustered nor intermingled with other tRNA genes. They occur as single copies within an approximately 415-bp repeat segment, which is separated from other initiator tRNA genes by a mean distance of 17 kb. In situ hybridization to polytene chromosomes localizes these genes to the 61D region of the Drosophila genome. Hybridization analysis of genomic DNA indicates the presence of 8-9 non-allelic initiator tRNA genes in Drosophila melanogaster.     

Mitochondrial sequence evolution in spiders: intraspecific variation in tRNAs lacking the TPsiC Arm

Analyses of mitochondrial DNA sequences from three species of Habronattus jumping spiders (Chelicerata: Arachnida: Araneae) reveal unusual inferred tRNA secondary structures and gene arrangements, providing new information on tRNA evolution within chelicerate arthropods. Sequences from the protein-coding genes NADH dehydrogenase subunit 1 (ND1), cytochrome oxidase subunit I (COI), and subunit II (COII) were obtained, along with tRNA, tRNA, and large-subunit ribosomal RNA (16S) sequences; these revealed several peculiar features. First, inferred secondary structures of tRNA and, likely, tRNA, lack the TPsiC arm and the variable arm and therefore do not form standard cloverleaf structures. In place of these arms is a 5-6-nt T arm-variable loop (TV) replacement loop such as that originally described from nematode mitochondrial tRNAs. Intraspecific variation occurs in the acceptor stem sequences in both tRNAs. Second, while the proposed secondary structure of the 3' end of 16S is similar to that reported for insects, the sequence at the 5' end is extremely divergent, and the entire gene is truncated about 300 nt with respect to Drosophila yakuba. Third, initiation codons appear to consist of ATY (ATT and ATC) and TTG for ND1 and COII, respectively. Finally, Habronattus shares the same ND1-tRNA-16S gene arrangement as insects and crustaceans, thus illustrating variation in a tRNA gene arrangement previously proposed as a character distinguishing chelicerates from insects and crustaceans.