Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.     

Expression Pattern Diversity and Functional Conservation Between Retroposed PRAT Genes from Drosophila melanogaster and Drosophila virilis

Gene duplication by retrotransposition duplicates only the coding and untranslated regions of a gene and, thus, biases retroduplicated genes toward having different expression patterns from their parental genes. As such, genes duplicated by retrotransposition are more likely to develop novel expression domains. To explore this idea further, we used the Prat/Prat2 gene duplication in Drosophila as a case study to examine the aftermath of a retrotransposition event that resulted in both the parent and the child gene becoming essential for survival. We used the Gal4-UAS transgene system with EGFP as a reporter to determine the developmental expression patterns of Prat and Prat2 from D. melanogaster (DmPrat and DmPrat2) and Prat from D. virilis (DvPrat). We also tested the functional equivalence of the protein products of DmPrat and DmPrat2. We found that each of the proteins could rescue DmPrat mutations, showing that the requirement for both Prat and Prat2 in Drosophila is not simply due to differences in protein function. In contrast, we found that the DmPrat and DmPrat2 genes have developed nonoverlapping patterns of expression, which correlate with their respective loss-of-function phenotypes. We further found that DvPrat expression is similar to DmPrat during development but differs in adult gonads. Thus, the function of the Prat retrogene has not diverged in the D. melanogaster and D. virilis lineages, while some aspects of its expression pattern have evolved. Finally, we have identified promoter elements, conserved upstream of DmPrat and DvPrat, that this retrogene has acquired to drive its expression.     

Characterization of Maltase Clusters in the Genus <Emphasis Type="Italic">Drosophila</Emphasis>

To reveal evolutionary history of maltase gene family in the genus Drosophila, we undertook a bioinformatics study of maltase genes from available genomes of 12 Drosophila species. Molecular evolution of a closely related glycoside hydrolase, the α-amylase, in Drosophila has been extensively studied for a long time. The α-amylases were even used as a model of evolution of multigene families. On the other hand, maltase, i.e., the α-glucosidase, got only scarce attention. In this study, we, therefore, investigated spatial organization of the maltase genes in Drosophila genomes, compared the amino acid sequences of the encoded enzymes and analyzed the intron/exon composition of orthologous genes. We found that the Drosophila maltases are more numerous than previously thought (ten instead of three genes) and are localized in two clusters on two chromosomes (2L and 2R). To elucidate the approximate time line of evolution of the clusters, we estimated the order and dated duplication of all the 10 genes. Both clusters are the result of ancient series of subsequent duplication events, which took place from 352 to 61 million years ago, i.e., well before speciation to extant Drosophila species. Also observed was a remarkable intron/exon composition diversity of particular maltase genes of these clusters, probably a result of independent intron loss after duplication of intron-rich gene ancestor, which emerged well before speciation in a common ancestor of all extant Drosophila species.     

Patterns of DNA-Sequence Divergence Between <Emphasis Type="Italic">Drosophila miranda</Emphasis> and <Emphasis Type="Italic">D. pseudoobscura</Emphasis>

Contrary to the classical view, a large amount of non-coding DNA seems to be selectively constrained in Drosophila and other species. Here, using Drosophila miranda BAC sequences and the Drosophila pseudoobscura genome sequence, we aligned coding and non-coding sequences between D. pseudoobscura and D. miranda, and investigated their patterns of evolution. We found two patterns that have previously been observed in comparisons between Drosophila melanogaster and its relatives. First, there is a negative correlation between intron divergence and intron length, suggesting that longer non-coding sequences may contain more regulatory elements than shorter sequences. Our other main finding is a negative correlation between the rate of non-synonymous substitutions (d _N) and codon usage bias (F _op), showing that fast-evolving genes have a lower codon usage bias, consistent with strong positive selection interfering with weak selection for codon usage.     

Evolution and arrangement of the <Emphasis Type="Italic">hsp70</Emphasis> gene cluster in two closely related species of the <Emphasis Type="Italic">virilis</Emphasis> group of <Emphasis Type="Italic">Drosophila</Emphasis>

To investigate the genetic basis of differing thermotolerance in the closely related species Drosophila virilis and Drosophila lummei, which replace one another along a latitudinal cline, we characterized the hsp70 gene cluster in multiple strains of both species. In both species, all hsp70 copies cluster in a single chromosomal locus, 29C1, and each cluster includes two hsp70 genes arranged as an inverted pair, the ancestral condition. The total number of hsp70 copies is maximally seven in the more thermotolerant D. virilis and five in the less tolerant D. lummei, with some strains of each species exhibiting lower copy numbers. Thus, maximum hsp70 copy number corresponds to hsp70 mRNA and Hsp70 protein levels reported previously and the size of heat-induced puffs at 29C1. The nucleotide sequence and spacing of the hsp70 copies are consistent with tandem duplication of the hsp70 genes in a common ancestor of D. virilis and D. lummei followed by loss of hsp70 genes in D. lummei. These and other data for hsp70 in Drosophila suggest that evolutionary adaptation has repeatedly modified hsp70 copy number by several different genetic mechanisms.     

Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

The regulation of <Emphasis Type="Italic">yp3</Emphasis> expression in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> fat body

The regulation of the Drosophila melanogaster yolk protein genes 1 and 2 have been well characterised. Cis-acting DNA elements and trans-acting factors regulating ovarian fat body and sex-specific expression have been identified. In this paper we have analysed the regulation of yolk protein 3, which is separated from the other two genes on the X-chromosome. We have separated sex-specific control from fat body control in some constructs in transgenic flies. We propose that the organisation of the regulatory elements in yp3 differs from yp1 and yp2 for control of fat body expression and that it closely resembles the regulation of a reporter gene using Musca and Calliphora yp promoter enhancer sequences in transgenic Drosophila.     

Comparative genomic analysis of CIPK gene family in <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Populus</Emphasis>

Calcium serves as a second messenger in various signal transduction pathways in plants. CBL-interacting protein kinases (CIPKs), which have a variety of functions, are involved in calcium signal transduction. Previous, the studies on CIPK family members focused on Arabidopsis and rice. Here, we present a comparative genomic analysis of the CIPK gene family in Arabidopsis and poplar, a model tree species. Twenty-seven potential CIPKs were identified from poplar using genome-wide analysis. Like the CIPK gene family from Arabidopsis, CIPK genes from poplar were also divided into intron-free and intron-harboring groups. In the intron-harboring group, the intron distribution of CIPKs is rather conserved during the genome evolutionary process. Many homologous gene pairs were found in the CIPK gene family, indicating duplication events might contribute to the amplification of this gene family. The phylogenetic comparison of CIPKs in combination with intron distribution analysis revealed that CIPK genes from both Arabidopsis and poplar might have an ancient origin, which formed earlier than the separation of these two eudicot species. Our genomic and bioinformatic analysis will provide an important foundation for further functional dissection of the CBL-CIPK signaling network in poplars. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Evolution of the <Emphasis Type="Italic">CPP-like</Emphasis> Gene Family in Plants: Insights from Comparative Genomics of <Emphasis Type="Italic">Arabidopsis</Emphasis> and Rice

CPP-like genes are members of a small family which features the existence of two similar Cys-rich domains termed CXC domains in their protein products and are distributed widely in plants and animals but do not exist in yeast. The members of this family in plants play an important role in development of reproductive tissue and control of cell division. To gain insights into how CPP-like genes evolved in plants, we conducted a comparative phylogenetic and molecular evolutionary analysis of the CPP-like gene family in Arabidopsis and rice. The results of phylogeny revealed that both gene loss and species-specific expansion contributed to the evolution of this family in Arabidopsis and rice. Both intron gain and intron loss were observed through intron/exon structure analysis for duplicated genes. Our results also suggested that positive selection was a major force during the evolution of CPP-like genes in plants, and most amino acid residues under positive selection were disproportionately located in the region outside the CXC domains. Further analysis revealed that two CXC domains and sequences connecting them might have coevolved during the long evolutionary period.     

The relationships among the species of the <Emphasis Type="Italic">Drosophila virilis</Emphasis> group inferred from the gene <Emphasis Type="Italic">Ras1</Emphasis> sequences

Comparative analysis of a group of closely related Drosophila species (D. virilis, D. lummei, D. novamexicana, D. americana texana, D. flavomontana, D. montana, D. borealis, D. lacicola, D. littoralis, D. kanekoi, and D. ezoana) was conducted based on an incomplete sequence of gene Ras1. The pattern of the relationships among the species corresponded to that expected from analysis of morphological and cytogenetic characters. Statistical data favoring neutrality of the substitutions examined in the Ras1 gene are presented. This character of the gene Ras1 evolution confers more reliability to reconstruction of phylogenetic relationships among closely related species. The resultant tree for main phylads of the group is as follows: D. virilis (D. lummei, D. montana, D. ezoana).     

Evidence for introgression in differentiated North-American and Finnish <Emphasis Type="Italic">Drosophila montana</Emphasis> populations

The virilis group species Drosophila montana is widely distributed around the northern hemisphere. Here we show that it consists of at least two well differentiated populations (Finnish and North-American populations) that have been diverging for the last 0.55–0.95 My. These populations show significant chromosomal, behavioural and morphological differences, but no apparent postzygotic isolation. Evidence for introgression is found for both Finnish and North-American populations at two out of the three X-linked genes (fused, elav and su(s)) studied here. In the light of these findings, previously reported evidence for selective sweeps in D. montana populations is re-evaluated.     

Molecular cloning and characterization of the <Emphasis Type="Italic">Dicer-like</Emphasis> 2 gene from <Emphasis Type="Italic">Brassica rapa</Emphasis>

Dicer-like proteins (DCLs) are involved in small RNA-mediated development and viral defense in plants. In model plants, at least four DCLs have been found and a number of studies have helped to understand their function. However, the function of the Dicer or DCLs in other plants is still unclear. Here, we report the full-length cDNA sequence of Brassica rapa ssp. chinensis DCL2 (BrDCL2) gene, which contains a 4,179 bp open reading frame (ORF) encoding a protein of 1,392 amino acids. At the 3′ end of BrDCL2, clones with three different lengths of 3′ untranslated region were found. An alternative splice variant of BrDCL2, BrDCL2sv, in which one intron was retained between exon9 and exon10, was also cloned. Because of a change in the coding sequence resulting in a premature terminal codon, BrDCL2sv was expected to translate a short peptide containing the whole DEXHc domain.     

Analysis of two promoters that control the expression of the <Emphasis Type="Italic">GTP cyclohydrolase I</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

GTP cyclohydrolase I (GTPCH) is a key enzyme in the de novo synthesis of tetrahydrobiopterin. Previously, the Drosophila melanogaster GTPCH gene has been shown to be expressed from two different promoters (P1 and P2). In our study, the 5′-flanking DNA regions required for P1 and P2 promoter activities were characterized using transient expression assay. The DNA regions between −98 and +31, and between −73 and +35 are required for efficient P1 and P2 promoter activities, respectively. The regions between −98 and −56 and between −73 and −41 may contain critical elements required for the expression of GTPCH in Drosophila. By aligning the nucleotide sequences in the P1 and P2 promoter regions of the Drosophila melanogaster and Drosophila virilis GTPCH genes, several conserved elements including palindromic sequences in the regions critical for P1 and P2 promoter activities were identified. Western blot analysis of transgenic flies transformed using P1 or P2 promoter-lacZ fusion plasmids further revealed that P1 promoter expression is restricted to the late pupae and adult developmental stages but that the P2 promoter driven expression of GTPCH is constitutive throughout fly development. In addition, X-gal staining of the embryos and imaginal discs of transgenic flies suggests that the P2 promoter is active from stage 13 of embryo and is generally active in most regions of the imaginal discs at the larval stages.     

Convergent intron loss of MRP1 in Drosophila and mosquito species

Previous study revealed that the MRP1 gene ortholog DMRP1/CG6214 of Drosophila melanogaster contains 12 exons in the coding region. In the current study, the genes of DMRP1/CG6214 from D. melanogaster and Drosophila virilis were compared, and the result indicated that D. virilis had an extra intron located in exon 2, implying that intron loss or gain might have occurred at this locus. To track the evolution of the extra intron (Intron Z), orthologous nucleotide sequences of 37 arthropod species were cloned or annotated. Based on phylogenetic analysis, we found that Intron Z should present in the common ancestor of arthropod species, more than 420 Ma. In addition, we found that Sophophora subgenus species and mosquito (Culex pipiens) lost Intron Z independently, showing evolutionary convergence.     

Gene Expression,Intron Density,and Splice Site Strength in <Emphasis Type="Italic">Drosophila</Emphasis> and <Emphasis Type="Italic">Caenorhabditis</Emphasis>

In this paper we investigate the relationships among intron density (number of introns per kilobase of coding sequence), gene expression level, and strength of splicing signals in two species: Drosophila melanogaster and Caenorhabditis elegans. We report a negative correlation between intron density and gene expression levels, opposite to the effect previously observed in human. An increase in splice site strength has been observed in long introns in D. melanogaster. We show this is also true of C. elegans. We also examine the relationship between intron density and splice site strength. There is an increase in splice site strength as the intron structure becomes less dense. This could suggest that introns are not recognized in isolation but could function in a cooperative manner to ensure proper splicing. This effect remains if we control for the effects of alternative splicing on splice site strength. Reviewing Editor: Dr. Nicolas Galtier