The cytochrome b and ATPase genes of honeybee mitochondrial DNA.

The gene sequences for honeybee cytochrome b, ATPase 6, and ATPase 8 are presented, along with the inferred amino acid sequences of the proteins. These mitochondrial genes are in the same relative positions as are their counterparts in Drosophila mitochondrial mtDNA and have evolved at a significantly greater overall rate than have those of Drosophila. Comparisons using both amino acid identity and the proportion of conservative replacements between the inferred Apis and vertebrate cytochrome b sequences shows the two highly conserved sections reported by Howell, but his recognition of five conserved regions is not well supported. A very high AT bias is reflected in very high codon biases. The best predictors of the number of occurrences of an amino acid in honeybee cytochrome b are the T and G contents of its codon family--unlike the case for vertebrate cytochrome b, in which the codon family size and AT bias are the strongest predictors; protein function, at least as judged by hydrophilicity characteristics, appears to be unaffected by these differing influences on amino acid composition.     

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.     

Heterogeneity in regional GC content and differential usage of codons and amino acids in GC-poor and GC-rich regions of the genome of Apis mellifera

The honeybee (Apis mellifera) has a genome with a wide variation in GC content showing 2 clear modal GC values, in some ways reminiscent of an isochore-like structure. To gain insight into causes and consequences of this pattern, we used a comparative approach to study the genome-wide alignment of primarily coding sequence of A. mellifera with Drosophila melanogaster and Anopheles gambiae. The latter 2 species show a higher average GC content than A. mellifera and no indications of bimodality, suggesting that the GC-poor mode is a derived condition in honeybee. In A. mellifera, synonymous sites of genes generally adopt the GC content of the region in which they reside. A large proportion of genes in GC-poor regions have not been assigned to the honeybee assembly because of the low sequence complexity of their genome neighborhood. The synonymous substitution rate between A. mellifera and the other species is very close to saturation, but analyses of nonsynonymous substitutions as well as amino acid substitutions indicate that the GC-poor regions are not evolving faster than the GC-rich regions. We describe the codon usage and amino acid usage and show that they are remarkably heterogeneous within the honeybee genome between the 2 different GC regions. Specifically, the genes located in GC-poor regions show a much larger deviation in both codon usage bias and amino acid usage from the Dipterans than the genes located in the GC-rich regions.     

The actin genes of Drosophila: protein coding regions are highly conserved but intron positions are not

The entire set of six closely related Drosophila actin genes was isolated using recombinant DNA methodology, and the structures of the respective coding regions were characterized by gene mapping techniques and by nucleotide sequencing of selected portions. Structural comparisons of these genes have resulted in several unexpected findings. Most striking is the nonconservation of the positions of intervening sequences within the protein-encoding regions of these genes. One of the Drosophila actin genes, DmA4, is split within a glycine codon at position 13; none of the remaining five genes is interrupted in the analogous position. Another gene, DmA6, is split within a glycine codon at position 307; at least two of the Drosophila actin genes are not split in the analogous position. Additionally, none of the Drosophila actin genes is split within codon four, where the yeast actin gene is interrupted. The six Drosophila actin genes encode several different proteins, but the amino acid sequence of each is similar to that of vertebrate cytoplasmic actins. None of the genes encodes a protein comparable in primary sequence to vertebrate skeletal muscle actin. Surprisingly, in each of these derived actin amino acid sequences in the initiator methionine is directly followed by a cysteine residue, which in turn precedes the string of three acidic amino acids characteristic of the amino termini of mature vertebrate cytoplasmic actins. We discuss these findings in the context of actin gene evolution and function.     

Correlations between the compositional properties of human genes,codon usage,and amino acid composition of proteins

Summary We have analyzed the correlation that exists between the GC levels of third and first or second codon position for about 1400 human coding sequences. The linear relationship that was found indicates that the large differences in GC level of third codon positions of human genes are paralleled by smaller differences in GC levels of first and second codon positions. Whereas third codon position differences correspond to very large differences in codon usage within the human genome, the first and second codon position differences correspond to smaller, yet very remarkable, differences in the amino acid composition of encoded proteins. Because GC levels of codon positions are linearly correlated with the GC levels of the isochores harboring the corresponding genes, both codon usage and amino acid composition are different for proteins encoded by genes located in isochores of different GC levels. Furthermore, we have also shown that a linear relationship with a unity slope and a correlation coefficient of 0.77 exists between GC levels of introns and exons from the 238 human genes currently available for this analysis. Introns are, however, about 5% lower in GC, on average, than exons from the same genes.     

The CO-I and CO-II region of honeybee mitochondrial DNA: evidence for variation in insect mitochondrial evolutionary rates

The sequence of a region of honeybee (Apis mellifera ligustica) mitochondrial DNA, which contains the genes for cytochrome c oxidase subunits I and II (CO-I and CO-II) and inferred genes for tRNA(Asp), tRNA(Leu)UUR, tRNA(Lys), and tRNA(Trp), is presented. The region includes the segment previously identified as incurring a length increase in some other bee strains, including Africanized bees. The sequence information of this study and of that by Vlasak et al. shows that several shifts of tRNA genes have occurred between Apis and Drosophila, but shifts of other kinds of genes have yet to be demonstrated. The CO-I and CO-II gene sequences are both more A+T rich than are the corresponding Drosophila genes. Parsimony analyses using the mouse and Xenopus sequences as outgroups show significantly more amino acid substitutions on the branch to Apis (120) than on that to Drosophila (44), indicating a difference in the long-term evolutionary rates of hymenopteran and dipteran mtDNA.     

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,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     

Unraveling Selection in the Mitochondrial Genome of Drosophila

We examine mitochondrial DNA variation at the cytochrome b locus within and between three species of Drosophila to determine whether patterns of variation conform to the predictions of neutral molecular evolution. The entire 1137-bp cytochrome b locus was sequenced in 16 lines of Drosophila melanogaster, 18 lines of Drosophila simulans and 13 lines of Drosophila yakuba. Patterns of variation depart from neutrality by several test criteria. Analysis of the evolutionary clock hypothesis shows unequal rates of change along D. simulans lineages. A comparison within and between species of the ratio of amino acid replacement change to synonymous change reveals a relative excess of amino acid replacement polymorphism compared to the neutral prediction, suggestive of slightly deleterious or diversifying selection. There is evidence for excess homozygosity in our world wide sample of D. melanogaster and D. simulans alleles, as well as a reduction in the number of segregating sites in D. simulans, indicative of selective sweeps. Furthermore, a test of neutrality for codon usage shows the direction of mutations at third positions differs among different topological regions of the gene tree. The analyses indicate that molecular variation and evolution of mtDNA are governed by many of the same selective forces that have been shown to govern nuclear genome evolution and suggest caution be taken in the use of mtDNA as a ``neutral' molecular marker.     

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.     

Variable strength of translational selection among 12 Drosophila species

Codon usage bias in Drosophila melanogaster genes has been attributed to negative selection of those codons whose cellular tRNA abundance restricts rates of mRNA translation. Previous studies, which involved limited numbers of genes, can now be compared against analyses of the entire gene complements of 12 Drosophila species whose genome sequences have become available. Using large numbers (6138) of orthologs represented in all 12 species, we establish that the codon preferences of more closely related species are better correlated. Differences between codon usage biases are attributed, in part, to changes in mutational biases. These biases are apparent from the strong correlation (r = 0.92, P < 0.001) among these genomes' intronic G + C contents and exonic G + C contents at degenerate third codon positions. To perform a cross-species comparison of selection on codon usage, while accounting for changes in mutational biases, we calibrated each genome in turn using the codon usage bias indices of highly expressed ribosomal protein genes. The strength of translational selection was predicted to have varied between species largely according to their phylogeny, with the D. melanogaster group species exhibiting the strongest degree of 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.     

ON COMBINING PROTEIN SEQUENCES AND NUCLEIC ACID SEQUENCES IN PHYLOGENETIC ANALYSIS: THE HOMEOBOX PROTEIN CASE

Abstract— Amino acid encoding genes contain character state information that may be useful for phylogenetic analysis on at least two levels. The nucleotide sequence and the translated amino acid sequences have both been employed separately as character states for cladistic studies of various taxa, including studies of the genealogy of genes in multigene families. In essence, amino acid sequences and nucleic acid sequences are two different ways of character coding the information in a gene. Silent positions in the nucleotide sequence (first or third positions in codons that can accrue change without changing the identity of the amino acid that the triplet codes for) may accrue change relatively rapidly and become saturated, losing the pattern of historical divergence. On the other hand, non-silent nucleotide alterations and their accompanying amino acid changes may evolve too slowly to reveal relationships among closely related taxa. In general, the dynamics of sequence change in silent and non-silent positions in protein coding genes result in homoplasy and lack of resolution, respectively. We suggest that the combination of nucleic acid and the translated amino acid coded character states into the same data matrix for phylogenetic analysis addresses some of the problems caused by the rapid change of silent nucleotide positions and overall slow rate of change of non-silent nucleotide positions and slowly changing amino acid positions. One major theoretical problem with this approach is the apparent non-independence of the two sources of characters. However, there are at least three possible outcomes when comparing protein coding nucleic acid sequences with their translated amino acids in a phylogenetic context on a codon by codon basis. First, the two character sets for a codon may be entirely congruent with respect to the information they convey about the relationships of a certain set of taxa. Second, one character set may display no information concerning a phylogenetic hypothesis while the other character set may impart information to a hypothesis. These two possibilities are cases of non-independence, however, we argue that congruence in such cases can be thought of as increasing the weight of the particular phylogenetic hypothesis that is supported by those characters. In the third case, the two sources of character information for a particular codon may be entirely incongruent with respect to phylogenetic hypotheses concerning the taxa examined. In this last case the two character sets are independent in that information from neither can predict the character states of the other. Examples of these possibilities are discussed and the general applicability of combining these two sources of information for protein coding genes is presented using sequences from the homeobox region of 46 homeobox genes fromDrosophila melanogasterto develop a hypothesis of genealogical relationship of these genes in this large multigene family.     

Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes

We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena. The codon usage of the L3 gene is highly biased. A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias. Class A comprises r-protein genes and a number of other highly expressed genes. Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1. Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases. Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions. The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena.     

Synonymous Codon Choices in the Extremely GC-Poor Genome of Plasmodium falciparum: Compositional Constraints and Translational Selection

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection. Received: 10 November 1998 / Accepted: 28 January 1999     

Amtyr1: characterization of a gene from honeybee (Apis mellifera) brain encoding a functional tyramine receptor

Biogenic amine receptors are involved in the regulation and modulation of various physiological and behavioral processes in both vertebrates and invertebrates. We have cloned a member of this gene family from the CNS of the honeybee, Apis mellifera. The deduced amino acid sequence is homologous to tyramine receptors cloned from Locusta migratoria and Drosophila melanogaster as well as to an octopamine receptor cloned from Heliothis virescens. Functional properties of the honeybee receptor were studied in stably transfected human embryonic kidney 293 cells. Tyramine reduced forskolin-induced cyclic AMP production in a dose-dependent manner with an EC50 of approximately 130 nM. A similar effect of tyramine was observed in membrane homogenates of honeybee brains. Octopamine also reduced cyclic AMP production in the transfected cell line but was both less potent (EC50 of approximately 3 microM) and less efficacious than tyramine. Receptor-encoding mRNA has a wide-spread distribution in the brain and subesophageal ganglion of the honeybee, suggesting that this tyramine receptor is involved in sensory signal processing as well as in higher-order brain functions.     

The Effects of Mutation and Natural Selection on Codon Bias in the Genes of Drosophila

Codon bias varies widely among the loci of Drosophila melanogaster, and some of this diversity has been explained by variation in the strength of natural selection. A study of correlations between intron and coding region base composition shows that variation in mutation pattern also contributes to codon bias variation. This finding is corroborated by an analysis of variance (ANOVA), which shows a tendency for introns from the same gene to be similar in base composition. The strength of base composition correlations between introns and codon third positions is greater for genes with low codon bias than for genes with high codon bias. This pattern can be explained by an overwhelming effect of natural selection, relative to mutation, in highly biased loci. In particular, this correlation is absent when examining fourfold degenerate sites of highly biased genes. In general, it appears that selection acts more strongly in choosing among fourfold degenerate codons than among twofold degenerate codons. Although the results indicate regional variation in mutational bias, no evidence is found for large scale regions of compositional homogeneity.