Delineating slowly and rapidly evolving fractions of the Drosophila genome.

Evolutionary conservation is an important indicator of function and a major component of bioinformatic methods to identify non-protein-coding genes. We present a new Bayesian method for segmenting pairwise alignments of eukaryotic genomes while simultaneously classifying segments into slowly and rapidly evolving fractions. We also describe an information criterion similar to the Akaike Information Criterion (AIC) for determining the number of classes. Working with pairwise alignments enables detection of differences in conservation patterns among closely related species. We analyzed three whole-genome and three partial-genome pairwise alignments among eight Drosophila species. Three distinct classes of conservation level were detected. Sequences comprising the most slowly evolving component were consistent across a range of species pairs, and constituted approximately 62-66% of the D. melanogaster genome. Almost all (>90%) of the aligned protein-coding sequence is in this fraction, suggesting much of it (comprising the majority of the Drosophila genome, including approximately 56% of non-protein-coding sequences) is functional. The size and content of the most rapidly evolving component was species dependent, and varied from 1.6% to 4.8%. This fraction is also enriched for protein-coding sequence (while containing significant amounts of non-protein-coding sequence), suggesting it is under positive selection. We also classified segments according to conservation and GC content simultaneously. This analysis identified numerous sub-classes of those identified on the basis of conservation alone, but was nevertheless consistent with that classification. Software, data, and results available at www.maths.qut.edu.au/-keithj/. Genomic segments comprising the conservation classes available in BED format.     

Putative microtubule-associated proteins from the Arabidopsis genome

Summary. Plant microtubule-associated proteins (MAPs) are important in modulating the function of the microtubule cytoskeleton. Various plant MAPs have already been described. However, because of the complexity of the plant microtubule cytoskeleton and its responses to developmental and environmental stimuli, there are undoubtedly many more MAPs to be discovered. We have used a literature search and the BLAST protein comparison program to identify which model MAPs from other taxa have close homologues in Arabidopsis thaliana. The search revealed Arabidopsis homologues of 14 model MAPs, with E values (numbers of proteins that will match the model protein merely by chance) of <1×10^–10 and homologous domains spanning 98–599 amino acid residues, representing 57.1–97.0% of the model MAP sequence, as well as 22.5–72.8% amino acid identities and 76.3–96.2% conservation of secondary structure in the homologous domain. All of the Arabidopsis homologues have either a full cDNA clone or an expressed sequence tag in the GenBank database and therefore are expressed. The proteins are likely to regulate a variety of functions, including tubulin folding, microtubule nucleation and polymerisation dynamics, microtubule-dependent cell cycle control, organisation of microtubule arrays, interaction of microtubules with plasma-membrane-associated protein complexes, and interactions with various other proteins. The exact functions of these putative MAPs in the plant cell remain to be elucidated empirically. The identification of these putative MAPs opens new avenues for the investigation of the complexities of the plant microtubule cytoskeleton.Present address: School of Biological Sciences, University of Manchester, Manchester, United Kingdom.Correspondence and reprints: School of Biological Sciences A12, University of Sydney, NSW 2006, Australia.Received October 21, 2002; accepted December 30, 2002; published online September 23, 2003     

Genes encoding calmodulin-binding proteins in the Arabidopsis genome.

Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.     

The evolving fungal genome

Fungal genomes vary considerably in size and organization. The genome of Microsporidium contains less than 3 Mb while the genomes of several Basidiomycetes and Ascomycetes greatly exceed 100 Mb. Likewise chromosome numbers and ploidy levels can differ even between closely related species. The differences in genome architecture among fungi reflect the interplay of different mutational processes as well as the population biology of the different species. Comparative genome studies have elucidated the underlying mechanisms of genome evolution in different groups of fungi and have provided insight into species-specific genomic traits. Mobile genetic elements have been instrumental in shaping the genome architecture and gene content in many fungal species. In many pathogenic fungi the mobile genetic elements even play a crucial role in rapid adaptive evolution by mediating high rates of sequence mutations, chromosomal rearrangements, and ploidy changes. But in many species mobile elements are efficiently restricted by defense mechanisms, which have evolved to suppress and regulate parasitic elements. Different rates of genome dynamic and adaptive evolution may reflect varying effective population sizes through which genetic drift and natural selection have differentially affected genome architecture in fungi over time.     

Mammalian sperm proteins are rapidly evolving: evidence of positive selection in functionally diverse genes

A growing number of genes involved in sex and reproduction have been demonstrated to be rapidly evolving. Here, we show that genes expressed solely in spermatozoa represent a highly diverged subset among mouse and human tissue-specific orthologs. The average rate of nonsynonymous substitutions per site (K(a)) is significantly higher in sperm proteins (mean K(a) = 0.18; N = 35) than in proteins expressed specifically in all other tissues (mean K(a) = 0.074; N = 473). No differences, however, are found in the synonymous substitution rate (K(s)) between tissues, suggesting that selective forces, and not mutation rate, explain the high rate of replacement substitutions in sperm proteins. Four out of 19 sperm-specific genes with characterized function demonstrated evidence of strong positive Darwinian selection, including a protein involved in gene regulation, Protamine-1 (PRM1), a protein involved in glycolysis, GAPDS, and two egg-binding proteins, Adam-2 precursor (ADAM2) and sperm-adhesion molecule-1 (SAM1). These results demonstrate the rapid evolution of sperm-specific genes and highlight the molecular action of sexual selection on a variety of characters involved in mammalian sperm function.     

Comparing the qualitatively different effects rapidly evolving and rapidly induced defences have on predator-prey interactions

Interspecific interactions depend not only on the population densities of the interacting species, but on their phenotypes as well. Phenotypic variation can be plastic or heritable and both mechanisms can drive phenotypic change at rates comparable to or faster than those of ecological dynamics (e.g. changes in population abundances or spatial distributions). In this study, we compare the effects rapidly induced and rapidly evolving defences have on community dynamics by considering the fast phenotypic change limit using fast-slow systems theory. Our approach allows us to study phenotypically plastic and evolving systems with one overarching theory, thus capturing the effects rapidly induced defences have on ecological dynamics and how those effects differ from the effects of evolving defences. Our results show that rapidly induced defences tend to stabilize community dynamics and that some behaviours observed in rapidly evolving systems cannot be produced by phenotypic plasticity.     

Phylogenetic analysis of the rapidly evolving SuUR gene in insects

Different genome regions differ in replication timing during the S phase. Late-replicating sequences are often underreplicated in the Drosophila salivary-gland polytene chromosomes. The SuUR gene, whose mutation changes the replication time of late-replicating regions in salivary-gland cells, has been identified in Drosophila melanogaster. The SUUR protein lacks homologs by a BLAST search, and only moderate homology is observed between its N-terminal end and chromatin-remodeling proteins of the SWI2/SNF2 family. The gene and the protein were analyzed in insects. Orthologs of the SuUR gene were found in all annotated Drosophila species. The number of amino acid substitutions in the SUUR protein proved to be extremely high, corresponding to that of rapidly evolving genes. Orthologs with low homology were found in mosquitoes Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. No orthologs of the SuUR gene were detected beyond Diptera.     

The rapidly evolving field of plant centromeres

Meiotic and mitotic chromosome segregation are highly conserved in eukaryotic organisms, yet centromeres--the chromosomal sites that mediate segregation--evolve extremely rapidly. Plant centromeres have DNA elements that are shared across species, yet they diverge rapidly through large- and small-scale changes. Over evolutionary time-scales, centromeres migrate to non-centromeric regions and, in plants, heterochromatic knobs can acquire centromere activity. Discerning the functional significance of these changes will require comparative analyses of closely related species. Combined with functional assays, continued efforts in plant genomics will uncover key DNA elements that allow centromeres to retain their role in chromosome segregation while allowing rapid evolution.     

Localized egg-cell expression of effector proteins for targeted modification of the Arabidopsis genome

Targeted modification of the genome is an important genetic tool, which can be achieved via homologous, non-homologous or site-specific recombination. Although numerous efforts have been made, such a tool does not exist for routine applications in plants. This work describes a simple and useful method for targeted mutagenesis or gene targeting, tailored to floral-dip transformation in Arabidopsis, by means of specific protein expression in the egg cell. Proteins stably or transiently expressed under the egg apparatus-specific enhancer (EASE) were successfully localized to the area of the egg cell. Moreover, a zinc-finger nuclease expressed under EASE induced targeted mutagenesis. Mutations obtained under EASE control corresponded to genetically independent events that took place specifically in the germline. In addition, RAD54 expression under EASE led to an approximately 10-fold increase in gene targeting efficiency, when compared with wild-type plants. EASE-controlled gene expression provides a method for the precise engineering of the Arabidopsis genome through temporally and spatially controlled protein expression. This system can be implemented as a useful method for basic research in Arabidopsis, as well as in the optimization of tools for targeted genetic modifications in crop plants.     

Selection of Arabidopsis genes encoding secreted and plasma membrane proteins

Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms. Systematic cloning of the genes encoding these proteins is therefore of general interest. An effective method of trapping signal sequences was first described by Tashiro et al. (1993), and a similar yet more efficient method was reported by Klein et al. (1996) and Jacobs et al. (1997). In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins. Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel. All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis. The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced. The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein. These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.     

A rapidly evolving genomic toolkit for Drosophila heterochromatin

Heterochromatin is the enigmatic eukaryotic genome compartment found mostly at telomeres and centromeres. Conventional approaches to sequence assembly and genetic manipulation fail in this highly repetitive, gene-sparse, and recombinationally silent DNA. In contrast, genetic and molecular analyses of euchromatin-encoded proteins that bind, remodel, and propagate heterochromatin have revealed its vital role in numerous cellular and evolutionary processes. Utilizing the 12 sequenced Drosophila genomes, Levine et al¹ took a phylogenomic approach to discover new such protein “surrogates” of heterochromatin function and evolution. This paper reported over 20 new members of what was traditionally believed to be a small and static Heterochromatin Protein 1 (HP1) gene family. The newly identified HP1 proteins are structurally diverse, lineage-restricted, and expressed primarily in the male germline. The birth and death of HP1 genes follows a “revolving door” pattern, where new HP1s appear to replace old HP1s. Here, we address alternative evolutionary models that drive this constant innovation.     

Evolution in the fast lane: rapidly evolving sex-related genes in Drosophila

A large portion of the annotated genes in Drosophila melanogaster show sex-biased expression, indicating that sex and reproduction-related genes (SRR genes) represent an appreciable component of the genome. Previous studies, in which subsets of genes were compared among few Drosophila species, have found that SRR genes exhibit unusual evolutionary patterns. Here, we have used the newly released genome sequences from 12 Drosophila species, coupled to a larger set of SRR genes, to comprehensively test the generality of these patterns. Among 2505 SRR genes examined, including ESTs with biased expression in reproductive tissues and genes characterized as involved in gametogenesis, we find that a relatively high proportion of SRR genes have experienced accelerated divergence throughout the genus Drosophila. Several testis-specific genes, male seminal fluid proteins (SFPs), and spermatogenesis genes show lineage-specific bursts of accelerated evolution and positive selection. SFP genes also show evidence of lineage-specific gene loss and/or gain. These results bring us closer to understanding the details of the evolutionary dynamics of SRR genes with respect to species divergence.     

Extensive duplication and reshuffling in the Arabidopsis genome

Systematic analysis of the Arabidopsis genome provides a basis for detailed studies of genome structure and evolution. Members of multigene families were mapped, and random sequence alignment was used to identify regions of extended similarity in the Arabidopsis genome. Detailed analysis showed that the number, order, and orientation of genes were conserved over large regions of the genome, revealing extensive duplication covering the majority of the known genomic sequence. Fine mapping analysis showed much rearrangement, resulting in a patchwork of duplicated regions that indicated deletion, insertion, tandem duplication, inversion, and reciprocal translocation. The implications of these observations for evolution of the Arabidopsis genome as well as their usefulness for analysis and annotation of the genomic sequence and in comparative genomics are discussed.     

Nuclear genes that encode mitochondrial proteins for DNA and RNA metabolism are clustered in the Arabidopsis genome

The plant mitochondrial genome is complex in structure, owing to a high degree of recombination activity that subdivides the genome and increases genetic variation. The replication activity of various portions of the mitochondrial genome appears to be nonuniform, providing the plant with an ability to modulate its mitochondrial genotype during development. These and other interesting features of the plant mitochondrial genome suggest that adaptive changes have occurred in DNA maintenance and transmission that will provide insight into unique aspects of plant mitochondrial biology and mitochondrial-chloroplast coevolution. A search in the Arabidopsis genome for genes involved in the regulation of mitochondrial DNA metabolism revealed a region of chromosome III that is unusually rich in genes for mitochondrial DNA and RNA maintenance. An apparently similar genetic linkage was observed in the rice genome. Several of the genes identified within the chromosome III interval appear to target the plastid or to be targeted dually to the mitochondria and the plastid, suggesting that the process of endosymbiosis likely is accompanied by an intimate coevolution of these two organelles for their genome maintenance functions.