Patterns of intron sequence evolution in Drosophila are dependent upon length and GC content

Background  

Introns comprise a large fraction of eukaryotic genomes, yet little is known about their functional significance. Regulatory elements have been mapped to some introns, though these are believed to account for only a small fraction of genome wide intronic DNA. No consistent patterns have emerged from studies that have investigated general levels of evolutionary constraint in introns.     

Loss of the bloom syndrome helicase increases DNA ligase 4-independent genome rearrangements and tumorigenesis in aging Drosophila

Background

The BLM DNA helicase plays a vital role in maintaining genome stability. Mutations in BLM cause Bloom syndrome, a rare disorder associated with cancer predisposition and premature aging. Humans and mice with blm mutations have increased frequencies of spontaneous mutagenesis, but the molecular basis of this increase is not well understood. In addition, the effect of aging on spontaneous mutagenesis in blm mutants has not been characterized. To address this, we used a lacZ reporter system in wild-type and several mutant strains of Drosophila melanogaster to analyze mechanisms of mutagenesis throughout their lifespan.

Results

Our data show that Drosophila lacking BLM have an elevated frequency of spontaneous genome rearrangements that increases with age. Although in normal flies most genome rearrangements occur through DNA ligase 4-dependent classical end joining, most rearrangements that accumulate during aging in blm mutants do not require DNA ligase 4, suggesting the influence of an alternative end-joining mechanism. Adult blm mutants also display reduced lifespan and ligase 4-independent enhanced tumorigenesis in mitotically active tissues.

Conclusions

These results suggest that Drosophila BLM suppresses error-prone alternative end-joining repair of DNA double-strand breaks that can result in genome instability and tumor formation during aging. In addition, since loss of BLM significantly affects lifespan and tumorigenesis, the data provide a link between error-prone end joining, genome rearrangements, and tumor formation in a model metazoan.     

Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes

Background

The availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal, spatial or quantitative aspects of mRNA expression. Using the nearly completed human genome sequence, we combined in silico and experimental approaches to define the complete human nuclear receptor (NR) set. This information was used to carry out a comparative genomic study of the NR superfamily.

Results

Our analysis of the human genome identified two novel NR sequences. Both these contained stop codons within the coding regions, indicating that both are pseudogenes. One (HNF4 γ-related) contained no introns and expressed no detectable mRNA, whereas the other (FXR-related) produced mRNA at relatively high levels in testis. If translated, the latter is predicted to encode a short, non-functional protein. Our analysis indicates that there are fewer than 50 functional human NRs, dramatically fewer than in Caenorhabditis elegans and about twice as many as in Drosophila. Using the complete human NR set we made comparisons with the NR sets of C. elegans and Drosophila. Searches for the >200 NRs unique to C. elegans revealed no human homologs. The comparative analysis also revealed a Drosophila member of NR subfamily NR3, confirming an ancient metazoan origin for this subfamily.

Conclusions

This work provides the basis for new insights into the evolution and functional relationships of NR superfamily members.     

Identification of a set of miRNAs differentially expressed in transiently TIA-depleted HeLa cells by genome-wide profiling

Background

In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established.

Results

Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof ¹ /+; mnk ^p6 /+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks.

Conclusion

mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway.     

EST analysis of Ostreococcus lucimarinus, the most compact eukaryotic genome, shows an excess of introns in highly expressed genes

Background

The genome of the pico-eukaryotic (bacterial-sized) prasinophyte green alga Ostreococcus lucimarinus has one of the highest gene densities known in eukaryotes, yet it contains many introns. Phylogenetic studies suggest this unusually compact genome (13.2 Mb) is an evolutionarily derived state among prasinophytes. The presence of introns in the highly reduced O. lucimarinus genome appears to be in opposition to simple explanations of genome evolution based on unidirectional tendencies, either neutral or selective. Therefore, patterns of intron retention in this species can potentially provide insights into the forces governing intron evolution.

Methodology/Principal Findings

Here we studied intron features and levels of expression in O. lucimarinus using expressed sequence tags (ESTs) to annotate the current genome assembly. ESTs were assembled into unigene clusters that were mapped back to the O. lucimarinus Build 2.0 assembly using BLAST and the level of gene expression was inferred from the number of ESTs in each cluster. We find a positive correlation between expression levels and both intron number (R = +0.0893, p = <0.0005) and intron density (number of introns/kb of CDS; R = +0.0753, p = <0.005).

Conclusions/Significance

In a species with a genome that has been recently subjected to a great reduction of non-coding DNA, these results imply the existence of selective/functional roles for introns that are principally detectable in highly expressed genes. In these cases, introns are likely maintained by balancing the selective forces favoring their maintenance with other mutational and/or selective forces acting on genome size.     

Protein Polymorphism Is Negatively Correlated with Conservation of Intronic Sequences and Complexity of Expression Patterns in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We report a significant negative correlation between nonsynonymous polymorphism and intron length in Drosophila melanogaster. This correlation is similar to that between protein divergence and intron length previously reported in Drosophila. We show that the relationship can be explained by the content of conserved noncoding sequences (CNS) within introns. In addition, genes with a high regulatory complexity and many genetic interactions also exhibit larger amounts of CNS within their introns and lower values of nonsynonymous polymorphism. The present study provides relevant evidence on the importance of intron content and expression patterns on the levels of coding polymorphism. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Dmitri Petrov]     

Sense-antisense gene overlap is probably a cause for retaining the few introns in <Emphasis Type="Italic">Giardia</Emphasis> genome and the implications

Background

It is widely accepted that the last eukaryotic common ancestor and early eukaryotes were intron-rich and intron loss dominated subsequent evolution, thus the presence of only very few introns in some modern eukaryotes must be the consequence of massive loss. But it is striking that few eukaryotes were found to have completely lost introns. Despite extensive research, the causes of massive intron losses remain elusive. Actually the reverse question -- how the few introns can be retained under the evolutionary selection pressure of intron loss -- is equally significant but was rarely studied, except that it was conjectured that the essential functions of some introns prevent their loss. The situation that extremely few (eight) spliceosome-mediated cis-spliced introns present in the relatively simple genome of Giardia lamblia provides an excellent opportunity to explore this question.

Results

Our investigation found three types of distribution patterns of the few introns in the intron-containing genes: ancient intron in ancient gene, later-evolved intron in ancient gene, and later-evolved intron in later-evolved gene, which can reflect to some extent the dynamic evolution of introns in Giardia. Without finding any special features or functional importance of these introns responsible for their retention, we noticed and experimentally verified that some intron-containing genes form sense-antisense gene pairs with transcribable genes on their complementary strands, and that the introns just reside in the overlapping regions.

Conclusions

In Giardia’s evolution, despite constant evolutionary selection pressure of intron loss, intron gain can still occur in both ancient and later-evolved genes, but only a few introns are retained; at least the evolutionary retention of some of the introns might not be due to the functional constraint of the introns themselves but the causes outside of introns, such as the constraints imposed by other genomic functional elements overlapping with the introns. These findings can not only provide some clues to find new genomic functional elements -- in the areas overlapping with introns, but suggest that “functional constraint” of introns may not be necessarily directly associated with intron loss and gain, and that the real functions are probably still outside of our current knowledge.

Reviewers

This article was reviewed by Mikhail Gelfand, Michael Gray, and Igor Rogozin.

     

Genomic analysis of the relationship between gene expression variation and DNA polymorphism in Drosophila simulans

Background

Understanding how DNA sequence polymorphism relates to variation in gene expression is essential to connecting genotypic differences with phenotypic differences among individuals. Addressing this question requires linking population genomic data with gene expression variation.

Results

Using whole genome expression data and recent light shotgun genome sequencing of six Drosophila simulans genotypes, we assessed the relationship between expression variation in males and females and nucleotide polymorphism across thousands of loci. By examining sequence polymorphism in gene features, such as untranslated regions and introns, we find that genes showing greater variation in gene expression between genotypes also have higher levels of sequence polymorphism in many gene features. Accordingly, X-linked genes, which have lower sequence polymorphism levels than autosomal genes, also show less expression variation than autosomal genes. We also find that sex-specifically expressed genes show higher local levels of polymorphism and divergence than both sex-biased and unbiased genes, and that they appear to have simpler regulatory regions.

Conclusion

The gene-feature-based analyses and the X-to-autosome comparisons suggest that sequence polymorphism in cis-acting elements is an important determinant of expression variation. However, this relationship varies among the different categories of sex-biased expression, and trans factors might contribute more to male-specific gene expression than cis effects. Our analysis of sex-specific gene expression also shows that female-specific genes have been overlooked in analyses that only point to male-biased genes as having unusual patterns of evolution and that studies of sexually dimorphic traits need to recognize that the relationship between genetic and expression variation at these traits is different from the genome as a whole.     

Comparison of the oxidative phosphorylation (OXPHOS) nuclear genes in the genomes of Drosophila melanogaster, Drosophila pseudoobscura and Anopheles gambiae

Background

In eukaryotic cells, oxidative phosphorylation (OXPHOS) uses the products of both nuclear and mitochondrial genes to generate cellular ATP. Interspecies comparative analysis of these genes, which appear to be under strong functional constraints, may shed light on the evolutionary mechanisms that act on a set of genes correlated by function and subcellular localization of their products.

Results

We have identified and annotated the Drosophila melanogaster, D. pseudoobscura and Anopheles gambiae orthologs of 78 nuclear genes encoding mitochondrial proteins involved in oxidative phosphorylation by a comparative analysis of their genomic sequences and organization. We have also identified 47 genes in these three dipteran species each of which shares significant sequence homology with one of the above-mentioned OXPHOS orthologs, and which are likely to have originated by duplication during evolution. Gene structure and intron length are essentially conserved in the three species, although gain or loss of introns is common in A. gambiae. In most tissues of D. melanogaster and A. gambiae the expression level of the duplicate gene is much lower than that of the original gene, and in D. melanogaster at least, its expression is almost always strongly testis-biased, in contrast to the soma-biased expression of the parent gene.

Conclusions

Quickly achieving an expression pattern different from the parent genes may be required for new OXPHOS gene duplicates to be maintained in the genome. This may be a general evolutionary mechanism for originating phenotypic changes that could lead to species differentiation.     

Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome

Background

It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.

Results

We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences.

Conclusions

Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.     

Genome-wide acceleration of protein evolution in flies (Diptera)

Background

The rate of molecular evolution varies widely between proteins, both within and among lineages. To what extent is this variation influenced by genome-wide, lineage-specific effects? To answer this question, we assess the rate variation between insect lineages for a large number of orthologous genes.

Results

When compared to the beetle Tribolium castaneum, we find that the stem lineage of flies and mosquitoes (Diptera) has experienced on average a 3-fold increase in the rate of evolution. Pairwise gene comparisons between Drosophila and Tribolium show a high correlation between evolutionary rates of orthologous proteins.

Conclusion

Gene specific divergence rates remain roughly constant over long evolutionary times, modulated by genome-wide, lineage-specific effects. Among the insects analysed so far, it appears that the Tribolium genes show the lowest rates of divergence. This has the practical consequence that homology searches for human genes yield significantly better matches in Tribolium than in Drosophila. We therefore suggest that Tribolium is better suited for comparisons between phyla than the widely employed dipterans.     

Singular value decomposition-based regression identifies activation of endogenous signaling pathways in vivo

Background

The physical organization and chromosomal localization of genes within genomes is known to play an important role in their function. Most genes arise by duplication and move along the genome by random shuffling of DNA segments. Higher order structuring of the genome occurs in eukaryotes, where groups of physically linked genes are co-expressed. However, the contribution of gene duplication to gene order has not been analyzed in detail, as it is believed that co-expression due to recent duplicates would obscure other domains of co-expression.

Results

We have catalogued ordered duplicated genes in Drosophila melanogaster, and found that one in five of all genes is organized as tandem arrays. Furthermore, among arrays that have been spatially conserved over longer periods than would be expected on the basis of random shuffling, a disproportionate number contain genes encoding developmental regulators. Using in situ gene expression data for more than half of the Drosophila genome, we find that genes in these conserved clusters are co-expressed to a much higher extent than other duplicated genes.

Conclusions

These results reveal the existence of functional constraints in insects that retain copies of genes encoding developmental and regulatory proteins as neighbors, allowing their co-expression. This co-expression may be the result of shared cis-regulatory elements or a shared need for a specific chromatin structure. Our results highlight the association between genome architecture and the gene regulatory networks involved in the construction of the body plan.     

The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome

Background

Chromosomal deletions are used extensively in Drosophila melanogaster genetics research. Deletion mapping is the primary method used for fine-scale gene localization. Effective and efficient deletion mapping requires both extensive genomic coverage and a high density of molecularly defined breakpoints across the genome.

Results

A large-scale resource development project at the Bloomington Drosophila Stock Center has improved the choice of deletions beyond that provided by previous projects. FLP-mediated recombination between FRT-bearing transposon insertions was used to generate deletions, because it is efficient and provides single-nucleotide resolution in planning deletion screens. The 793 deletions generated pushed coverage of the euchromatic genome to 98.4%. Gaps in coverage contain haplolethal and haplosterile genes, but the sizes of these gaps were minimized by flanking these genes as closely as possible with deletions. In improving coverage, a complete inventory of haplolethal and haplosterile genes was generated and extensive information on other haploinsufficient genes was compiled. To aid mapping experiments, a subset of deletions was organized into a Deficiency Kit to provide maximal coverage efficiently. To improve the resolution of deletion mapping, screens were planned to distribute deletion breakpoints evenly across the genome. The median chromosomal interval between breakpoints now contains only nine genes and 377 intervals contain only single genes.

Conclusions

Drosophila melanogaster now has the most extensive genomic deletion coverage and breakpoint subdivision as well as the most comprehensive inventory of haploinsufficient genes of any multicellular organism. The improved selection of chromosomal deletion strains will be useful to nearly all Drosophila researchers.