Extensive gene amplification and concerted evolution within the CPR family of cuticular proteins in mosquitoes

Annotation of the Anopheles gambiae genome has revealed a large increase in the number of genes encoding cuticular proteins with the Rebers and Riddiford Consensus (the CPR gene family) relative to Drosophila melanogaster. This increase reflects an expansion of the RR-2 group of CPR genes, particularly the amplification of sets of highly similar paralogs. Patterns of nucleotide variation indicate that extensive concerted evolution is occurring within these clusters. The pattern of concerted evolution is complex, however, as sequence similarity within clusters is uncorrelated with gene order and orientation, and no comparable clusters occur within similarly compact arrays of the RR-1 group in mosquitoes or in either group in D. melanogaster. The dearth of pseudogenes suggests that sequence clusters are maintained by selection for high gene-copy number, perhaps due to selection for high expression rates. This hypothesis is consistent with the apparently parallel evolution of compact gene architectures within sequence clusters relative to single-copy genes. We show that RR-2 proteins from sequence-cluster genes have complex repeats and extreme amino-acid compositions relative to single-copy CPR proteins in An. gambiae, and that the amino-acid composition of the N-terminal and C-terminal sequence flanking the chitin-binding consensus region evolves in a correlated fashion.     

Special evolutionary properties of genes encoding a protein with a simple amino acid repeat

We have examined the evolution of a gene, SM50, encoding a component of the spicule matrix, which plays an integral role in the formation of the echinoderm skeleton. This gene was originally characterized in Strongylocentrotus purpuratus and encodes an imperfect tandem repeat of six or seven amino acids. We have analyzed the sequence of this repeat in a number of sea urchin species and have determined that the repeat regions have undergone concerted evolution. There are differences in the repeat region between species, but the overall repeat structure is conserved, suggesting the repeat forms a structural domain important in biomineralization. The inherent conserved amino acid repeat structure promotes concerted evolution due to the high probability of misreplication and unequal crossing-over in the repeated segment of the gene. While there are constraints on the amino acids allowed in the repeat region, there are also variations, so that the sequences observed illustrate the balance between amino acid substitutions and concerted evolution. We have evidence that substitutions can alter the mechanisms of unequal crossing-over, altering the way concerted evolution occurs. The way in which concerted evolution occurred appears to be determined by the degree of sequence similarity between the repeats in a given gene, which influences how unequal crossing over may occur. We have mapped the differences in repeat regions on existing phylogenetic trees and indicate where concerted evolution has taken place. We also confirm an earlier report that Hemicentrotus pulcherrimus fits into the Strongylocentrotus genus and examine the evolution of the H. pulcherrimus SM50 repeat relative to other members of this genus. Received: 31 October 2000 / Accepted: 20 March 2001     

Complete nucleotide sequence of the A+T-rich region of Drosophila mauritiana mitochondrial DNA

We determined the complete nucleotide sequence of the A+T-rich region of the maII type of mtDNA in D. mauritiana. The nucleotide sequence was found to contain 3,206 bp. Three types of conserved element, i.e., type I element, type II element, and T-stretch, were included in this sequence, as reported for D. melanogaster. Comparison between the two species revealed that the type I elements were less conserved than the type II elements. However, each of these type I elements contained a G-stretch within a loop of a putative stem-loop-forming sequence, which has also been observed in D. melanogaster. Moreover, in both type I and type II repeat arrays, the elements closest to the T-stretch diverged the most, due to nucleotide substitution and/or the insertion of short repeats. Sequence comparison of the two complete sequences of the A+T-rich region of D. melanogaster and the maII type of D. mauritiana, as well as comparison of partial sequences in other types of mtDNA within the melanogaster complex, suggested that the A+T-rich region in this complex has been maintained by concerted evolution after the duplication of two types of element, i.e., type I and type II.     

Divergence and heterogeneity of the histone gene repeating units in the Drosophila melanogaster species subgroup

The repeating units of the histone gene cluster containing the H1, H2A, H2B and H4 genes were amplified by PCR from the Drosophila melanogaster species subgroup, i.e., D. yakuba, D. erecta, D. sechellia, D. mauritiana, D. teissieri and D. orena. The PCR products were cloned and their nucleotide sequences of about 4.6-4.8kbp were determined to elucidate the mechanism of molecular evolution of the histone gene family. The heterogeneity among the histone gene repeating units was 0.6% and 0.7% for D. yakuba and D. sechellia, respectively, indicating the same level of heterogeneity as in the H3 gene region of D. melanogaster. Divergence of the genes among species even in the most closely related ones was much greater than the heterogeneity among family members, indicating a concerted mode of evolution for the histone gene repeating units. Among the species in the D. melanogaster species subgroup, the histone gene regions as well as 3rd codon position of the coding region showed nearly the same GC contents. These results suggested that the previous conclusion on analysis of the H3 gene regions, the gene family evolution in a concerted fashion, holds true for the whole histone gene repeating unit.     

The involucrin gene of the galago. Existence of a correction process acting on its segment of repeats

The involucrin gene of the galago, a prosimian, has been cloned and sequenced. The coding region contains a segment of repeats homologous to the segment of repeats in the gene of another prosimian, the lemur, and different from the segments of repeats in the genes of higher primates. The repeats lengths in the two prosimians are similar; and except for a single duplication of a block of repeats in the lemur alone, the number of repeats is the same. However, the nucleotide consensus sequences of the repeats differ between the two species at 3 out of 39 nucleotide positions. The repeats therefore appear to have been modified by a correction process that led toward homogeneity in the repeats of each species while permitting divergence between the two species. The correction process, an example of concerted evolution, has taken place preferentially between adjacent repeats. The numerous differences between the segments of repeats of higher primates and the segments of repeats of lower animals reveal a discontinuity in the evolutionary processes acting on the gene.     

Evolutionary conservation of the chromosomal configuration and regulation of amylase genes among eight species of the Drosophila melanogaster species subgroup

Nuclear DNA was extracted from each of the eight species comprising the Drosophila melanogaster species subgroup. Southern hybridization of this DNA by using a molecular probe specific for the alpha-amylase coding region showed that the duplicated structure of the amylase locus, first found in D. melanogaster, is conserved among all species of the melanogaster subgroup. Evidence is also presented for the concerted evolution of the duplicated genes within each species. In addition, it is shown that the glucose repression of amylase gene expression, which has been extensively studied in D. melanogaster, is not confined to this species but occurs in all eight members of the species subgroup. Thus, both the duplicated gene structure and the glucose repression of Drosophila amylase gene activity are stable over extended periods of evolutionary time.      

Adaptive evolution of Cid, a centromere-specific histone in Drosophila

Centromeric DNA is generally composed of large blocks of tandem satellite repeats that change rapidly due to loss of old arrays and expansion of new repeat classes. This extreme heterogeneity of centromeric DNA is difficult to reconcile with the conservation of the eukaryotic chromosome segregation machinery. Histone H3-like proteins, including Cid in Drosophila melanogaster, are a unique chromatin component of centromeres. In comparisons between closely related species of Drosophila, we find an excess of replacement changes that have been fixed since the separation of D. melanogaster and D. simulans, suggesting adaptive evolution. The last adaptive changes appear to have occurred recently, as evident from a reduction in polymorphism in the melanogaster lineage. Adaptive evolution has occurred both in the long N-terminal tail as well as in the histone fold of Cid. In the histone fold, the replacement changes have occurred in the region proposed to mediate binding to DNA. We propose that this rapid evolution of Cid is driven by a response to the changing satellite repeats at centromeres. Thus, centromeric H3-like proteins may act as adaptors between evolutionarily labile centromeric DNA and the conserved kinetochore machinery.     

Isolation and characterization of a Drosophila neuropeptide gene

We have purified a 9 amino acid amidated neuropeptide, DPKQDFMRFamide, from whole adult D. melanogaster. This peptide exhibits sequence homology to the molluscan bioactive tetrapeptide FMRFamide and is a novel member of the FMRFamide peptide family. The gene encoding DPKQDFMRFamide has been cloned and characterized. It is present in a single copy per haploid genome, is expressed as a unique 1.7 kb mRNA species, and cytologically maps to 46C on the right arm of chromosome 2. Characterization of a cDNA clone indicates that the precursor protein is 347 amino acids in length and contains 5 copies of DPKQDFMRFamide, as well as 10 additional amidated peptides exhibiting varying degrees of structural relatedness. The Drosophila DPKQDFMRFamide gene and the Aplysia FMRFamide gene are ancestrally related; however, peptides display a higher degree of homology within a species than between species, suggesting intragenic concerted evolution of these neuropeptides.     

The period gene of Drosophila carries species-specific behavioral instructions.

We have analyzed and compared the circadian locomotor activity rhythms of Drosophila melanogaster and D.pseudoobscura. The rhythms of D.pseudoobscura are stronger and the periods shorter than those of D.melanogaster. We have also transformed D.melanogaster flies with a hybrid gene containing the coding region of the D.pseudoobscura period (per) gene. Behavioral assays of flies containing this hybrid gene show that the per protein encoded by the D.pseudoobscura per gene is able to rescue the rhythmic deficiencies of arrhythmic, pero1 D.melanogaster. More important, the rhythms of some of these strains are stronger and the periods shorter than those of D.melanogaster (and those of transformants which carry the equivalent D.melanogaster per gene construct) and hence resemble those of D.pseudoobscura. The results suggest that the primary amino acid sequence of the per gene encodes species-specific behavioral instructions that are detectable when only the per gene is transferred to a different species.     

Evolutionary rearrangement of the amylase genomic regions between Drosophila melanogaster and Drosophila pseudoobscura

Two Drosophila pseudoobscura genomic clones have sequence similarity to the Drosophila melanogaster amylase region that maps to the 53CD region on the D. melanogaster cytogenetic map. The two clones with similarity to amylase map to sections 73A and 78C of the D. pseudoobscura third chromosome cytogenetic map. The complete sequences of both the 73A and 78C regions were compared to the D. melanogaster genome to determine if the coding region for amylase is present in both regions and to determine the evolutionary mechanism responsible for the observed distribution of the amylase gene or genes. The D. pseudoobscura 73A and 78C linkage groups are conserved with the D. melanogaster 41E and 53CD regions, respectively. The amylase gene, however, has not maintained its conserved linkage between the two species. These data indicate that amylase has moved via a transposition event in the D. melanogaster or D. pseudoobscura lineage. The predicted genes within the 73A and 78C regions show patterns of molecular evolution in synonymous and nonsynonymous sites that are consistent with previous studies of these two species.     

Turnover of R1 (Type I) and R2 (Type Ii) Retrotransposable Elements in the Ribosomal DNA of Drosophila Melanogaster

R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (less than 0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster.     

Stability of Tandem Repeats in the Drosophila Melanogaster HSR-Omega Nuclear RNA

The Drosophila melanogaster Hsr-omega locus produces a nuclear RNA containing >5 kb of tandem repeat sequences. These repeats are unique to Hsr-omega and show concerted evolution similar to that seen with classical satellite DNAs. In D. melanogaster the monomer is ~280 bp. Sequences of 191/2 monomers differ by 8 +/- 5% (mean +/- SD), when all pairwise comparisons are considered. Differences are single nucleotide substitutions and 1-3 nucleotide deletions/insertions. Changes appear to be randomly distributed over the repeat unit. Outer repeats do not show the decrease in monomer homogeneity that might be expected if homogeneity is maintained by recombination. However, just outside the last complete repeat at each end, there are a few fragments of sequence similar to the monomer. The sequences in these flanking regions are not those predicted for sequences decaying in the absence of recombination. Instead, the fragmentation of the sequence homology suggests that flanking regions have undergone more severe disruptions, possibly during an insertion or amplification event. Hsr-omega alleles differing in the number of repeats are detected and appear to be stable over a few thousand generations; however, both increases and decreases in repeat numbers have been observed. The new alleles appear to be as stable as their predecessors. No alleles of less than ~5 kb nor more than ~16 kb of repeats were seen in any stocks examined. The evidence that there is a limit on the minimum number of repeats is consistent with the suggestion that these repeats are important in the function of the unusual Hsr-omega nuclear RNA.     

Molecular Evolution of the Duplicated Amy Locus in the Drosophila Melanogaster Species Subgroup: Concerted Evolution Only in the Coding Region and an Excess of Nonsynonymous Substitutions in Speciation

From the analysis of restriction maps of the Amy region in eight sibling species belonging to the Drosophila melanogaster species subgroup, we herein show that the patterns of duplication of the Amy gene are almost the same in all species. This indicates that duplication occurred before speciation within this species subgroup. From the nucleotide sequence data, we show a strong within-species similarity between the duplicated loci in the Amy coding region. This is in contrast to a strong similarity in the 5' and 3' flanking regions within each locus (proximal or distal) throughout the species subgroup. This means that concerted evolution occurred only in the Amy coding region and that differentiated evolution between the duplication occurred in the flanking regions. Moreover, when comparing the species, we also found a significant excess of nonsynonymous substitutions. In particular, all the fixed substitutions specific to D. erecta were found to be nonsynonymous. We thus conclude that adaptive protein evolution occurred in the lineage of D. erecta that is a ``specialist' species for host plants and probably also occurs in the process of speciation in general.     

The Rate of Unequal Crossing Over in the dumpy Gene from Drosophila melanogaster

The PIGSFEAST (PF) exon of the Drosophila dumpy gene is undergoing concerted evolution by the process of unequal crossing over. We have developed a long-range PCR-based assay to amplify the approximately 12 kb long exon which contains variable numbers of 303 or 306 nt long repeats in a tandem array. We applied this procedure to mutation accumulation lines of Drosophila melanogaster established by M. Wayne and L. Higgins. Nine new repeat length variants were found in these lines allowing us to measure the rate of unequal crossing over in the PF exon. The rate, which for several reasons is an underestimate, is 7.05 × 10⁻⁴ exchanges per generation.     

Patterns of nucleotide polymorphism and divergence in the odorant-binding protein genes OS-E and OS-F: analysis in the melanogaster species subgroup of Drosophila

The Olfactory Specific-E and -F genes (OS-E and OS-F) belong to the odorant-binding protein gene family, which includes the general odorant-binding proteins and the pheromone-binding proteins. In Drosophila melanogaster, these genes are arranged in tandem in a genomic region near the centromere of chromosome arm 3R. We examined the pattern of DNA sequence variation in an approximately 7-kb genomic region encompassing the two OS genes in four species of the melanogaster subgroup of Drosophila and in a population sample of D. melanogaster. We found that both the OS-E and the OS-F gene are present in all surveyed species. Nucleotide divergence estimates would support that the two genes are functional, although they diverge in their functional constraint. The pattern of nucleotide variation in D. melanogaster also differed between genes. Variation in the OS-E gene region exhibited an unusual and distinctive pattern: (i) a relatively high number of fixed amino acid replacements in the encoded protein and (ii) a peak of nucleotide polymorphism around the OS-E gene. These results are unlikely under the neutral model and suggest the action of natural selection in the evolution of the two odorant-binding protein genes.