Comparative Analysis of Evolution in a Rodent Histone H2a Pseudogene

Sequences were obtained from five species of rodents that are orthologous to an H2a histone pseudogene from Mus musculus. The pseudogene is part of the cluster of replication-dependent histone genes found on Mus musculus chromosome 13. Comparative analysis of these five sequences together with the previously published sequence from M. musculus shows that this gene has likely been a pseudogene throughout the evolution of the genus Mus, while the gene from Rattus norvegicus is likely functional. Three large (>20 bp) deletions were found among the Mus pseudogenes, a feature that is very unusual compared to surveys of processed pseudogenes. In addition, there are two single-base deletions and one 4-bp insertion among the Mus pseudogenes. The species distributions of one of the large deletions and the 4-bp insertion require either independent insertions of an identical sequence, independent deletions with identical boundaries, or a deletion followed by precise reintegration of the original sequence. The evidence favors the hypothesis of multiple deletions with identical boundaries. The ``coding' regions of the Mus pseudogenes show a much reduced level of among-species variability in the 3′ half of the pseudogene, compared both to the 5′ half and to flanking sequences. This supports a hypothesis that the 3′ end of the pseudogene is the target of frequent gene conversion by functional H2a genes. Received: 1 April 1997 / Accepted: 12 June 1997     

Age and Detection of Retroprocessed Pseudogenes in Murine Rodents

Retroprocessed pseudogenes, calmodulin II (ψ₁, ψ₂, and ψ₃ CALMII), ψα-tubulin, π-glutathione S-transferase (ψπ-GST) from rat, lactic acid dehydrogenase (ψ LDH) from mouse, and heat shock protein 60 chaperonin (ψ HSP60) from Chinese hamster, were examined for their presence in these species by polymerase chain reaction (PCR). Pseudogenes of these murine rodents were detected by PCR only in those species in which the genes were originally identified, suggesting that the selected pseudogene of one species arose too recently to be detected in the genomes of the other rodent species. The calculated ages of the rodent pseudogenes ranged from 1.7 Myr (ψα-tubulin) to 7.5 Myr (ψ₃ CALMII) when employing a homologous functional gene of the taxon as a reference in the relative rate test with the mouse or rat as the outgroup. Given the high rate of divergence of the genes of rodents relative to other species, selection of an outgroup with similar mutation rates seems warranted. To justify further the conclusion that the selected pseudogenes were indeed retroprocessed after these three taxa diverged, the presence of the pseudogenes in the genome of different rat species was examined. The existence of ψ₃ CALMII and ψα-tubulin pseudogenes of Rattus norvegicus among species belonging to Rattus sensu stricto is evidence for the common ancestry of this group. Received: 23 May 2001 / Accepted: 31 May 2001     

A Translocated Mitochondrial Cytochrome b Pseudogene in Voles (Rodentia: Microtus)

A full-length cytochrome b pseudogene was found in rodents; it has apparently been translocated from a mitochondrion to the nuclear genome in the subfamily Arvicolinae. The pseudogene (ψcytb) differed from its mitochondrial counterpart at 201 of 1143 sites (17.6%) and by four indels. Cumulative evidence suggests that the pseudogene has been translocated to the nucleus. Phylogenetic reconstruction indicates that the pseudogene arose before the diversification of M. arvalis/M. rossiaemeridionalis from M. oeconomus, but after the divergence of the peromyscine/sigmodontine/arvicoline clades some ∼10 MYA. Published rates of divergence between mitochondrial genes and their nuclear pseudogenes suggest that the translocation of this mitochondrial gene to the nuclear genome occurred some 6 MYA, in agreement with the phylogenetic evidence. Received: 16 January 1998 / Accepted: 18 July 1998     

Identification of Ψ3Tom20, a novel processed pseudogene of the human Tom20 gene, and complete characterization of Ψ1Tom20 and Ψ2Tom20

We report the identification and characterization of Ψ3Tom20, a novel processed pseudogene of the human Tom20 (hTom20) gene, which is 96.2% similarity with the hTom20 cDNA and is 5′ and 3′ truncated. In addition, we present the complete characterization of Ψ1Tom20 and Ψ2Tom20, the two other recently reported members of this pseudogene family. Comparison of the sequences of Ψ3Tom20 with that of the previously reported Ψ2Tom20 revealed and corrected an error in the previously determined sequence of Ψ2Tom20. A detailed analysis of these three pseudogenes, including their flanking regions, is presented. It suggests they probably arose from mRNAs that were polyadenylated at different sites. Possible mechanisms involved in their integration as retroposons are also discussed. Received: 29 October 1998 / Accepted: 7 May 1999     

Heterogeneity in Codon Usage in the Flatworm Schistosoma mansoni

Synonymous codon choices vary considerably among Schistosoma mansoni genes. Principal components analysis detects a single major trend among genes, which highly correlates with GC content in third codon positions and exons, but does not discriminate among putatively highly and lowly expressed genes. The effective number of codons used in each gene, and its distribution when plotted against GC₃, suggests that codon usage is shaped mainly by mutational biases. The GC content of exons, GC₃, 5′, 3′, and flanking (5′+ 3′+ introns) regions are all correlated among them, suggesting that variations in GC content may exist among different regions of the S. mansoni genome. We propose that this genome structure might be among the most important factors shaping codon usage in this species, although the action of selection on certain sequences cannot be excluded. Received: 10 March 1997 / Accepted: 27 June 1997     

Dynamics and Phylogenetic Implications of MtDNA Control Region Sequences in New World Jays (Aves: Corvidae)

To study the evolution of mtDNA and the intergeneric relationships of New World Jays (Aves: Corvidae), we sequenced the entire mitochondrial DNA control region (CR) from 21 species representing all genera of New World jays, an Old World jay, crows, and a magpie. Using maximum likelihood methods, we found that both the transition/transversion ratio (κ) and among site rate variation (α) were higher in flanking domains I and II than in the conserved central domain and that the frequency of indels was highest in domain II. Estimates of κ and α were much more influenced by the density of taxon sampling than by alternative optimal tree topologies. We implemented a successive approximation method incorporating these parameters into phylogenetic analysis. In addition we compared our study in detail to a previous study using cytochrome b and morphology to examine the effect of taxon sampling, evolutionary rates of genes, and combined data on tree resolution. We found that the particular weighting scheme used had no effect on tree topology and little effect on tree robustness. Taxon sampling had a significant effect on tree robustness but little effect on the topology of the best tree. The CR data set differed nonsignificantly from the tree derived from the cytochrome b/morphological data set primarily in the placement of the genus Gymnorhinus, which is near the base of the CR tree. However, contrary to conventional taxonomy, the CR data set suggested that blue and black jays (Cyanocorax sensu lato) might be paraphyletic and that the brown jay Psilorhinus (=Cyanocorax) morio is the sister group to magpie jays (Calocitta), a phylogenetic hypothesis that is likely as parsimonious with regard to nonmolecular characters as monophyly of Cyanocorax. The CR tree also suggests that the common ancestor of NWJs was likely a cooperative breeder. Consistent with recent systematic theory, our data suggest that DNA sequences with high substitution rates such as the CR may nonetheless be useful in reconstructing relatively deep phylogenetic nodes in avian groups. Received: 10 November 1999 / Accepted: 16 March 2000     

Duplication and divergence of the genes of the α-esterase cluster ofDrosophila melanogaster

The α-esterase cluster of D. melanogaster contains 11 esterase genes dispersed over 60 kb. Embedded in the cluster are two unrelated open reading frames that have sequence similarity with genes encoding ubiquitin-conjugating enzyme and tropomyosin. The esterase amino acid sequences show 37–66% identity with one another and all but one have all the motifs characteristic of functional members of the carboxyl/cholinesterase multigene family. The exception has several frameshift mutations and appears to be a pseudogene. Patterns of amino acid differences among cluster members in relation to generic models of carboxyl/cholinesterase protein structure are broadly similar to those among other carboxyl/cholinesterases sequenced to date. However the α-esterases differ from most other members of the family in: their lack of a signal peptide; the lack of conservation in cysteines involved in disulfide bridges; and in four indels, two of which occur in or adjacent to regions that align with proposed substrate-binding sites of other carboxyl/cholinesterases. Phylogenetic analyses clearly identify three simple gene duplication events within the cluster. The most recent event involved the pseudogene which is located in an intron of another esterase gene. However, relative rate tests suggest that the pseudogene remained functional after the duplication event and has become inactive relatively recently. The distribution of indels also suggests a deeper node in the gene phylogeny that separates six genes at the two ends of the cluster from a block of five in the middle. Received: 18 January 1996 / Accepted: 12 March 1996     

Evolutionary Studies on Uricases of Fungal Endosymbionts of Aphids and Planthoppers

Aphids belonging to the three genera Tuberaphis, Glyphinaphis, and Cerataphis contain extracellular fungal symbionts that resemble endocellular yeast-like symbionts of planthoppers. Whereas the symbiont of planthoppers has a uricase (urate oxidase; EC 1.7.3.3) and recycles uric acid that the host stores, no uric acid was found in Tuberaphis styraci, and its fungal symbiont did not exhibit the uricase activity. However, the fungal symbionts of these aphids, including that of T. styraci, were shown to have putative uricase genes, or pseudogenes, for the uricase. Sequence analysis of these genes revealed that deleterious mutations occurred independently on each lineage of Glyphinaphis and Tuberaphis, while no such mutation was found in the lineage of Cerataphis. These genes were almost identical to those cloned from the symbionts of planthoppers, though the host aphids and planthoppers are phylogenetically distant. To estimate the phylogenetic relationship in detail between the fungal symbionts of aphids and those of planthoppers, a gene tree was constructed based on the sequences of the uricase genes including their flanking regions. As a result, the symbionts of planthoppers and Tuberaphis aphids formed a sister group against those of Glyphinaphis and Cerataphis aphids with high bootstrap confidence levels, which strongly suggests that symbionts have been horizontally transferred from the aphids' lineage to the planthoppers'. Received: 29 March 2000 / Accepted: 31 May 2000     

Male-Biased Mutation Rates Revealed from Z and W Chromosome-Linked ATP Synthase α-Subunit (ATP5A1) Sequences in Birds

Whether the mutation rate differs between sexes has been a matter of discussion for years. Molecular analyses of mammals have indicated that males mutate more often than females, as manifested by the faster rate of neutral sequence evolution on the Y chromosome than on the X chromosome. However, these observations can as well be interpreted as specific reduction of the X chromosome mutation rate, which would be adaptive because of reducing the number of slightly deleterious recessive mutations exposed in hemizygote males. Recently, data from birds have suggested that vertebrate mutation rates may indeed be male-biased. In birds, females are the heterogametic sex (ZW), and analyses of the Z-linked CHD1Z gene have shown that it evolves faster than its W-linked and thus female-specific homologue, CHD1W. We have now studied the second avian gene known to exist in a copy on the nonrecombining regions of both the Z and the W chromosome, viz., the ATP synthase α-subunit (ATP5A1). In independent comparisons of three pairs of bird species from divergent lineages, intron sequences of the Z-linked copy (ATP5A1Z) were consistently found to evolve faster than the W-linked copy (ATP5A1W). From these data we calculated male-to-female mutation rate ratios (α) of 1.8, 2.3, and 5.0 in Galliform, Anseriform, and Ciconiiform lineages, respectively. Therefore, this study provides independent support for a male-biased mutation rate in birds. Received: 15 July 1999 / Accepted: 5 January 2000     

A Complex Organization of the Gene Encoding Cytochrome Oxidase Subunit 1 in the Mitochondrial Genome of the Dinoflagellate, Crypthecodinium cohnii: Homologous Recombination Generates Two Different cox1 Open Reading Frames

In the course of investigating mitochondrial genome organization in Crypthecodinium cohnii, a non-photosynthetic dinoflagellate, we identified four EcoRI fragments that hybridize to a probe specific for cox1, the gene that encodes subunit 1 of cytochrome oxidase. Cloning and sequence characterization of the four fragments (5.7, 5.1, 4.1, 3.5 kilobase pairs) revealed that cox1 exists in four distinct but related contexts in C. cohnii mtDNA, with a central repeat unit flanked by one of two possible upstream (flanking domain 1 or 2) and downstream (flanking domain 3 or 4) regions. The majority of the cox1 gene is located within the central repeat; however, the C-terminal portion of the open reading frame extends into flanking domains 3 and 4, thereby creating two distinct cox1 coding sequences. The 3′-terminal region of one of the cox1 reading frames can assume an elaborate secondary structure, which potentially could act to stabilize the mature mRNA against nucleolytic degradation. In addition, a high density of small inverted repeats (15–22 base pairs) has been identified at the 5′-end of cox1, further suggesting that hairpin structures could be important for gene regulation. The organization of cox1 in C. cohnii mtDNA appears to reflect homologous recombination events within the central repeat between different cox1 sequence contexts. Such recombining repeats are a characteristic feature of plant (angiosperm) mtDNA, but they have not previously been described in the mitochondrial genomes of protists. Received: 21 December 2000 / Accepted: 30 January 2001     

Multiple Origins of Cyclodiene Insecticide Resistance in Tribolium castaneum (Coleoptera: Tenebrionidae)

The number of origins of pesticide resistance-associated mutations is important not only to our understanding of the evolution of resistance but also in modeling its spread. Previous studies of amplified esterase genes in a highly dispersive Culex mosquito have suggested that insecticide resistance-associated mutations (specifically a single-gene duplication event) can occur a single time and then spread throughout global populations. In order to provide data for resistance-associated point mutations, which are more typical of pesticide mechanisms as a whole, we studied the number of independent origins of cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum. Target-site insensitivity to cyclodienes is conferred by single point mutations in the gene Resistance to dieldrin (Rdl), which codes for a subunit of a γ-aminobutyric acid (GABA) receptor. These point mutations are associated with replacements of alanine 302 which render the receptor insensitive to block by the insecticide. We collected 141 strains of Tribolium worldwide and screened them for resistance. Twenty-four strains contained resistant individuals. After homozygosing 23 of these resistance alleles we derived a nucleotide sequence phylogeny of the resistant strains from a 694-bp section of Rdl, encompassing exon 7 (which contains the resistance-associated mutation) and part of a flanking intron. The phylogeny also included six susceptible alleles chosen at random from a range of geographical locations. Resistance alleles fell into six clades and three clades contained both resistant and susceptible alleles. Although statistical analysis provided support at only the 5–6% level, the pattern of variation in resistance alleles is more readily explained by multiple independent origins of resistance than by spread of a single resistance-associated mutation. For example, two resistance alleles differed from two susceptible alleles only by the resistance-associated mutation itself, suggesting that they form the susceptible ancestors and that resistance arose independently in several susceptible backgrounds. This suggests that in Tribolium Rdl, de novo mutations for resistance have arisen independently in several populations. Identical alleles were found in geographically distant regions as well, also implying that some Rdl alleles have been exported in stored grain. These differences from the Culex study may stem both from differences in the population genetics of Tribolium versus that of mosquitoes and differences in mutation rates associated with point mutations versus gene duplication events. The Tribolium data therefore suggest that multiple origins of insecticide resistance (associated with specific point mutations) may be more common than the spread of single events. These findings have implications for the way in which we model the evolution and spread of insecticide resistance genes and also suggest that parallel adaptive substitutions may not be uncommon in phyletic evolution. Received: 14 October 1998 / Accepted: 4 January 1999     

Tail-to-Head Arrangement of a Partial Chicken Glyceraldehyde-3-Phosphate Dehydrogenase Processed Pseudogene

A chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) processed pseudogene was identified by inverse PCR using oligonucleotide primers specific for the 5′ region of the GAPDH mRNA. Molecular cloning and sequence analysis of this genomic sequence shows that the processed pseudogene is incomplete and arranged in a permuted tail-to-head order. We propose that the tail-to-head organization is the result of circularization and breakage of a GAPDH retrogene prior to chromosomal integration. PCR analysis of DNAs from quail, pheasant, and various jungle fowl, shows that the processed pseudogene was formed after the three genera diverged but prior to Gallus speciation. This is the first report of a chicken GAPDH processed pseudogene sequence. This is also the first published report of a processed pseudogene with a tail-to-head organization. Received: 15 November 1996 / Accepted: 1 April 1997     

The Old World sparrows (genus Passer) phylogeography and their relative abundance of nuclear mtDNA pseudogenes

The phylogenetic relationships of genus Passer (Old World sparrows) have been studied with species covering their complete world living range. Mitochondrial (mt) cyt b genes and pseudogenes have been analyzed, the latter being strikingly abundant in genus Passer compared with other studied songbirds. The significance of these Passer pseudogenes is presently unclear. The mechanisms by which mt cyt b genes become pseudogenes after nuclear translocation are discussed together with their mode of evolution, i.e., transition/transversion mitochondrial ratio is decreased in the nucleus, as is the constraint for variability at the three codon positions. However, the skewed base composition according to codon position (in 1^st position the percentage is very similar for the four bases, in 2^nd position there are fewer percentage of A and G and more percentage of T, and in 3^rd codon position fewer percentage of G and T and is very rich in A and C) is maintained in the translocated nuclear pseudogenes. Different nuclear internal mechanisms and/or selective pressures must exist for explaining this nuclear/mitochondrial differential DNA base evolutive variability. Also, the phylogenetic usefulness of pseudogenes for defining relationships between closely related lineages is stressed. The analyses suggest that the primitive genus Passer species comes from Africa, the Cape sparrow being the oldest: P. hispaniolensis italiae is more likely conspecific to P. domesticus than to P. hispaniolensis. Also, Passer species are not included within weavers or Estrildinae or Emberizinae, as previously suggested. European and American Emberizinae sparrows are closely related to each other and seem to be the earliest species that radiated among the studied songbirds (all in the Miocene Epoch). Received: 29 November 2000 / Accepted: 22 March 2001     

The BC200 RNA Gene and Its Neural Expression Are Conserved in Anthropoidea (Primates)

The gene encoding BC200 RNA arose from a monomeric Alu element. Subsequently, the RNA had been recruited or exapted into a function of the nervous system. Here we confirm the presence of the BC200 gene in several primate species among the Anthropoidea. The period following the divergence of New World monkeys and Old World monkeys from their common ancestor is characterized by a significantly higher substitution rate in the examined 5′ flanking region than in the BC200 RNA coding region itself. Furthermore, the conservation of CpG dimers in the RNA coding region (200 bp) is drastically increased compared to the 5′ flanking region (∼400 bp) over all 12 species examined. Finally, the brain-specific expression pattern of BC200 RNA and its presence as a ribonucleoprotein particle (RNP) are conserved in Old World and New World monkeys. Our studies indicate that the gene encoding BC200 RNA was created at least 35–55 million years ago and its presence, mode of expression, and association with protein(s) as an RNP are under selective pressure. Received: 1 December 1997 / Accepted: 3 June 1998     

Are Noncoding Sequences of Rickettsia prowazekii Remnants of ``Neutralized' Genes?

It has been hypothesized that a large fraction of 24% noncoding DNA in R. prowazekii consists of degraded genes. This hypothesis has been based on the relatively high G+C content of noncoding DNA. However, a comparison with other genomes also having a low overall G+C content shows that this argument would also apply to other bacteria. To test this hypothesis, we study the coding potential in sets of genes, pseudogenes, and intergenic regions. We find that the correlation function and the χ²-measure are clearly indicative of the coding function of genes and pseudogenes. However, both coding potentials make almost no indication of a preexisting reading frame in the remaining 23% of noncoding DNA. We simulate the degradation of genes due to single-nucleotide substitutions and insertions/deletions and quantify the number of mutations required to remove indications of the reading frame. We discuss a reduced selection pressure as another possible origin of this comparatively large fraction of noncoding sequences. Received: 27 December 1999 / Accepted: 5 July 2000     

Evolution of Growth Hormone in Primates: The GH Gene Clusters of the New World Monkeys Marmoset (Callithrix jacchus) and White-Fronted Capuchin (Cebus albifrons)

The GH gene cluster in marmoset, Callithrix jacchus, comprises eight GH-like genes and pseudogenes and appears to have arisen as a consequence of gene duplications occurring independently of those leading to the human GH gene cluster. We report here the complete sequence of the marmoset GH gene locus, including the intergenic regions and 5′ and 3′ flanking sequence, and a study of the multiple GH-like genes of an additional New World monkey (NWM), the white-fronted capuchin, Cebus albifrons. The marmoset sequence includes 945 nucleotides (nt) of 5′ flanking sequence and 1596 nt of 3′ flanking sequence that are “unique”; between these are eight repeat units, including the eight GH genes/pseudogenes. The breakpoints between these repeats are very similar, indicating a regular pattern of gene duplication. These breakpoints do not correspond to those found in the much less regular human GH gene cluster. This and phylogenetic analysis of the repeat units within the marmoset gene cluster strongly support the independent origin of these gene clusters, and the idea that the episode of rapid evolution that occurred during GH evolution in primates preceded the gene duplications. The marmoset GH gene cluster also differs from that of human in having fewer and more evenly distributed Alu sequences (a single pair in each repeat unit) and a “P-element” upstream of every gene/pseudogene. In human there is no P-element upstream of the gene encoding pituitary GH, and these elements have been implicated in placental expression of the other genes of the cluster. The GH gene clusters in marmoset and capuchin appear to have arisen as the consequence of a single-gene duplication event, but in capuchin there was then a remarkable expansion of the GH locus, giving at least 40 GH-like genes and pseudogenes. Thus even among NWMs the GH gene cluster is very variable. [Reviewing Editor: Nicolas Galtier]     

Characteristic Sequence Pattern in the 5- to 20-bp Upstream Region of Primate Alu Elements

We conducted comprehensive sequence analysis of 5′ flanking regions of primate Alu elements. Information contents were computed and frequencies of 1024 pentanucleotides were measured to approximate the location of a characteristic sequence and to specify its pattern(s), which may be involved in the integration of Alu elements into their host genomes. A large number of samples was used, the wide region of the 5′ end of Alu elements was analyzed, and comparisons were made among different subfamilies. Through our analyses, ``TTTTAAAAA' or ``(T) _m (A) _n ' can be stated as a candidate for the characteristic sequence pattern, which resides around the region 5 to 20 base pairs upstream of the 5′ end of Alu elements. This characteristic sequence pattern was more prominent in the sequences of younger Alus, which is a strong indication that the sequence pattern has a role at the time of Alu integration. Received: 10 May 1999 / Accepted: 1 October 1999     

Compositional Correlations in the Chicken Genome

This paper analyses the compositional correlations that hold in the chicken genome. Significant linear correlations were found among the regions studied—coding sequences (and their first, second, and third codon positions), flanking regions (5′ and 3′), and introns—as is the case in the human genome. We found that these compositional correlations are not limited to global GC levels but even extend to individual bases. Furthermore, an analysis of 1037 coding sequences has confirmed a correlation among GC₃, GC₂, and GC₁. The implications of these results are discussed. Received: 9 December 1998 / Accepted: 18 April 1999     

Fluctuating Mutation Bias and the Evolution of Base Composition in Drosophila

The idea that the pattern of point mutation in Drosophila has remained constant during the evolution of the genus has recently been challenged. A study of the nucleotide composition focused on the Drosophila saltans group has evidenced unsuspected nucleotide composition differences among lineages. Compositional differences are associated with an accelerated rate of amino acid replacement in functionally less constrained regions. Here we reassess this issue from a different perspective. Adopting a maximum-likelihood estimation approach, we focus on the different predictions that mutation and selection make about the nonsynonymous-to-synonymous rate ratio. We investigate two gene regions, alcohol dehydrogenase (Adh) and xanthine dehydrogenase (Xdh), using a balanced data set that comprises representatives from the melangaster, obscura, saltans, and willistoni groups. We also consider representatives of the Hawaiian picture-winged group. These Hawaiian species are known to have experienced repeated bottlenecks and are included as a reference for comparison. Our results confirm patterns previously detected. The branch ancestral to the fast-evolving willistoni/saltans lineage, where most of the change in GC content has occurred, exhibits an excess of synonymous substitutions. The shift in mutation bias has affected the extent of the rate variation among sites in Xdh. Received: 4 May 1999 / Accepted: 26 July 1999