Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species.

MAIN CONCLUSION: 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

Diversity and dynamics of the Drosophila transcriptome.

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.

Population-genomic insights into the evolutionary origin and fate of obligately asexual Daphnia pulex.

Despite much theoretical work, the molecular-genetic causes and evolutionary consequences of asexuality remain largely undetermined. Asexual animal species are rare, evolutionarily short-lived, and thought to suffer mutational meltdown as a result of lack of recombination. Whole-genome analysis of 11 sexual and 11 asexual genotypes of Daphnia pulex indicates that current asexual lineages are in fact very young, exhibit no signs of purifying selection against accumulating mutations, and have extremely high rates of gene conversion and deletion. The reconstruction of chromosomal haplotypes in regions containing SNP markers associated with asexuality (chromosomes VIII and IX) indicates that introgression from a sister species, Daphnia pulicaria, underlies the origin of the asexual phenotype. Silent-site divergence of the shared chromosomal haplotypes of asexuals indicates that the spread of asexuality is as recent as 1,250 y, although the origin of the meiosis-suppressing element or elements could be substantially older. In addition, using previous estimates of the gene conversion rate from Daphnia mutation accumulation lines, we are able to age each asexual lineage. Although asexual lineages originate from wide crosses that introduce elevated individual heterozygosities on clone foundation, they also appear to be constrained by the inbreeding-like effect of loss of heterozygosity that accrues as gene conversion and hemizygous deletion expose preexisting recessive deleterious alleles of asexuals, limiting their evolutionary longevity. Our study implies that the buildup of newly introduced deleterious mutations (i.e., Muller's ratchet) may not be the dominant force imperiling nonrecombining populations of D. pulex, as previously proposed.

Global patterns of tissue-specific alternative polyadenylation in Drosophila.

We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3' untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3' UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3' UTR length in the nervous system.

The spread of a transposon insertion in Rec8 is associated with obligate asexuality in Daphnia.

Although transitions from sexual to asexual reproduction are thought to have important evolutionary consequences, little is known about the mechanistic underpinnings of these changes. The cyclical parthenogen Daphnia pulex is a powerful model in which to address these issues because female-limited meiosis suppression can be transmitted to sexual individuals via males, providing the opportunity for genetic dissection of the trait. A previous study identified genomic regions differentiating obligately asexual females from their sexual counterparts, and a candidate gene within one such region, encoding the meiotic cohesin Rec8, is the subject of this investigation. The D. pulex genome contains three Rec8 loci, all of which are quite polymorphic. However, at one of the loci, all obligately asexual clones carry an allele containing an identical upstream insertion of a transposable element as well as a frameshift mutation, both of which are completely absent from sexual lineages. The low level of variation within the insertion allele across all asexual lineages suggests that this element may be in the process of spreading through the species, and abrogation or modification of Rec8 function is possibly responsible for converting meiotically reproducing lineages into obligate asexuals.

The ecoresponsive genome of Daphnia pulex.

We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabases and contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of gene duplication resulting in tandem gene clusters. More than a third of Daphnia's genes have no detectable homologs in any other available proteome, and the most amplified gene families are specific to the Daphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests that the maintenance of duplicated genes is not random, and the analysis of gene expression under different environmental conditions reveals that numerous paralogs acquire divergent expression patterns soon after duplication. Daphnia-specific genes, including many additional loci within sequenced regions that are otherwise devoid of annotations, are the most responsive genes to ecological challenges.

The transcriptional diversity of 25 Drosophila cell lines.

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.

Gene networks in Drosophila melanogaster: integrating experimental data to predict gene function.

BACKGROUND: Discovering the functions of all genes is a central goal of contemporary biomedical research. Despite considerable effort, we are still far from achieving this goal in any metazoan organism. Collectively, the growing body of high-throughput functional genomics data provides evidence of gene function, but remains difficult to interpret. RESULTS: We constructed the first network of functional relationships for Drosophila melanogaster by integrating most of the available, comprehensive sets of genetic interaction, protein-protein interaction, and microarray expression data. The complete integrated network covers 85% of the currently known genes, which we refined to a high confidence network that includes 20,000 functional relationships among 5,021 genes. An analysis of the network revealed a remarkable concordance with prior knowledge. Using the network, we were able to infer a set of high-confidence Gene Ontology biological process annotations on 483 of the roughly 5,000 previously unannotated genes. We also show that this approach is a means of inferring annotations on a class of genes that cannot be annotated based solely on sequence similarity. Lastly, we demonstrate the utility of the network through reanalyzing gene expression data to both discover clusters of coregulated genes and compile a list of candidate genes related to specific biological processes. CONCLUSIONS: Here we present the the first genome-wide functional gene network in D. melanogaster. The network enables the exploration, mining, and reanalysis of experimental data, as well as the interpretation of new data. The inferred annotations provide testable hypotheses of previously uncharacterized genes.

Methods for meiotic chromosome preparation, immunofluorescence, and fluorescence in situ hybridization in Daphnia pulex.

The genus Daphnia has an intriguing reproductive mode of cyclical parthenogenesis. This reproductive mode has been studied for centuries, but cytogenetic information is lacking due to technical limitations of classical methods. We have developed methods for the preparation and examination of meiotic chromosomes of Daphnia pulex from oocytes and spermatocytes. Oocyte chromosome preparations are obtained by isolating individual oocytes after the release of yolk granules from the ovary using pressure and capillary action. Spermatocyte chromosomes are prepared using a conventional squash method. Cryosectioning is an easy and fast way to prepare sections. We also illustrate the application of immunofluorescence staining against alpha tubulin, as well as fluorescence in situ hybridization (FISH) using the intergenic spacer of ribosomal DNA or single-copy cosmid clones.

Meiosis genes in Daphnia pulex and the role of parthenogenesis in genome evolution.

BACKGROUND: Thousands of parthenogenetic animal species have been described and cytogenetic manifestations of this reproductive mode are well known. However, little is understood about the molecular determinants of parthenogenesis. The Daphnia pulex genome must contain the molecular machinery for different reproductive modes: sexual (both male and female meiosis) and parthenogenetic (which is either cyclical or obligate). This feature makes D. pulex an ideal model to investigate the genetic basis of parthenogenesis and its consequences for gene and genome evolution. Here we describe the inventory of meiotic genes and their expression patterns during meiotic and parthenogenetic reproduction to help address whether parthenogenesis uses existing meiotic and mitotic machinery, or whether novel processes may be involved. RESULTS: We report an inventory of 130 homologs representing over 40 genes encoding proteins with diverse roles in meiotic processes in the genome of D. pulex. Many genes involved in cell cycle regulation and sister chromatid cohesion are characterized by expansions in copy number. In contrast, most genes involved in DNA replication and homologous recombination are present as single copies. Notably, RECQ2 (which suppresses homologous recombination) is present in multiple copies while DMC1 is the only gene in our inventory that is absent in the Daphnia genome. Expression patterns for 44 gene copies were similar during meiosis versus parthenogenesis, although several genes displayed marked differences in expression level in germline and somatic tissues. CONCLUSION: We propose that expansions in meiotic gene families in D. pulex may be associated with parthenogenesis. Taking into account our findings, we provide a mechanistic model of parthenogenesis, highlighting steps that must differ from meiosis including sister chromatid cohesion and kinetochore attachment.

Recent advances in crustacean genomics.

Crustaceans are a diverse and ancient group of arthropods that have long been studied as interesting model systems in biology, especially for understanding animal evolution and physiology and for environmentally relevant studies. Like many model systems, advances in DNA-sequencing methodologies have led to a large amount of genomics-related projects. The purpose of this article is to highlight the genome projects and functional genomics (transcriptomics) projects that are currently underway in crustacean biology. Specifically, we have surveyed the amount of publicly available DNA sequence data (both genomic and EST data) across all crustacean taxa for which a significant number of DNA sequences have been generated. Several ongoing projects are presented including the ecology of invasive species, thermal physiology, ion and water balance, ecology and evolutionary biology, and developmental biology.

Profiling sex-biased gene expression during parthenogenetic reproduction in Daphnia pulex.

BACKGROUND: Sexual reproduction is a core biological function that is conserved throughout eukaryotic evolution, yet breeding systems are extremely variable. Genome-wide comparative studies can be effectively used to identify genes and regulatory patterns that are constrained to preserve core functions from those that may help to account for the diversity of animal reproductive strategies. We use a custom microarray to investigate gene expression in males and two reproductive stages of females in the crustacean Daphnia pulex. Most Daphnia species reproduce by cyclical parthenogenesis, alternating between sexual and clonal reproduction. Both sex determination and the switch in their mode of reproduction is environmentally induced, making Daphnia an interesting comparative system for the study of sex-biased and reproductive genes. RESULTS: Patterns of gene expression in females and males reveal that 50% of assayed transcripts show some degree of sex-bias. Female-biased transcription is enriched for translation, metabolic and regulatory genes associated with development. Male-biased expression is enriched for cuticle and protease function. Comparison with well studied arthropods such as Drosophila melanogaster and Anopheles gambiae suggests that female-biased patterns tend to be conserved, whereas male-biased genes are evolving faster in D. pulex. These findings are based on the proportion of female-biased, male-biased, and unbiased genes that share sequence similarity with proteins in other animal genomes. CONCLUSION: Some transcriptional differences between males and females appear to be conserved across Arthropoda, including the rapid evolution of male-biased genes which is observed in insects and now in a crustacean. Yet, novel patterns of male-biased gene expression are also uncovered. This study is an important first step towards a detailed understanding of the genetic basis and evolution of parthenogenesis, environmental sex determination, and adaptation to aquatic environments.

Sampling Daphnia's expressed genes: preservation, expansion and invention of crustacean genes with reference to insect genomes.

BACKGROUND: Functional and comparative studies of insect genomes have shed light on the complement of genes, which in part, account for shared morphologies, developmental programs and life-histories. Contrasting the gene inventories of insects to those of the nematodes provides insight into the genomic changes responsible for their diversification. However, nematodes have weak relationships to insects, as each belongs to separate animal phyla. A better outgroup to distinguish lineage specific novelties would include other members of Arthropoda. For example, crustaceans are close allies to the insects (together forming Pancrustacea) and their fascinating aquatic lifestyle provides an important comparison for understanding the genetic basis of adaptations to life on land versus life in water. RESULTS: This study reports on the first characterization of cDNA libraries and sequences for the model crustacean Daphnia pulex. We analyzed 1,546 ESTs of which 1,414 represent approximately 787 nuclear genes, by measuring their sequence similarities with insect and nematode proteomes. The provisional annotation of genes is supported by expression data from microarray studies described in companion papers. Loci expected to be shared between crustaceans and insects because of their mutual biological features are identified, including genes for reproduction, regulation and cellular processes. We identify genes that are likely derived within Pancrustacea or lost within the nematodes. Moreover, lineage specific gene family expansions are identified, which suggest certain biological demands associated with their ecological setting. In particular, up to seven distinct ferritin loci are found in Daphnia compared to three in most insects. Finally, a substantial fraction of the sampled gene transcripts shares no sequence similarity with those from other arthropods. Genes functioning during development and reproduction are comparatively well conserved between crustaceans and insects. By contrast, genes that were responsive to environmental conditions (metal stress) and not sex-biased included the greatest proportion of genes with no matches to insect proteomes. CONCLUSION: This study along with associated microarray experiments are the initial steps in a coordinated effort by the Daphnia Genomics Consortium to build the necessary genomic platform needed to discover genes that account for the phenotypic diversity within the genus and to gain new insights into crustacean biology. This effort will soon include the first crustacean genome sequence.

Mitochondrial mRNA stability and polyadenylation during anoxia-induced quiescence in the brine shrimp Artemia franciscana.

Polyadenylation of messenger RNA is known to be an important mechanism for regulating mRNA stability in a variety of systems, including bacteria, chloroplasts and plant mitochondria. By comparison, little is known about the role played by polyadenylation in animal mitochondrial gene expression. We have used embryos of the brine shrimp Artemia franciscana to test hypotheses regarding message stability and polyadenylation under conditions simulating anoxia-induced quiescence. In response to anoxia, these embryos undergo a profound and acute metabolic downregulation, characterized by a steep drop in intracellular pH (pH(i)) and ATP levels. Using dot blots of total mitochondrial RNA, we show that during in organello incubations both O(2) deprivation and acidic pH (pH 6.4) elicit increases in half-lives of selected mitochondrial transcripts on the order of five- to tenfold or more, relative to normoxic controls at pH 7.8. Polyadenylation of these transcripts was measured under the same incubation conditions using a reverse transcriptase-polymerase chain reaction (RT-PCR)-based assay. The results demonstrate that low pH and anoxia promote significant deadenylation of the stabilized transcripts in several cases, measured either as change over time in the amount of polyadenylation within a given size class of poly(A)(+) tail, or as the total amount of polyadenylation at the endpoint of the incubation. This study is the first direct demonstration that for a metazoan mitochondrion, polyadenylation is associated with destabilized mRNA. This pattern has also been demonstrated in bacteria, chloroplasts and plant mitochondria and may indicate a conserved mechanism for regulating message half-life that differs from the paradigm for eukaryotic cytoplasm, where increased mRNA stability is associated with polyadenylation.

Transcriptional initiation under conditions of anoxia-induced quiescence in mitochondria from Artemia franciscana embryos.

In response to anoxia, embryos of the brine shrimp Artemia franciscana are able coordinately to downregulate metabolism to levels low enough to permit survival for several years at room temperature. In addition to dramatic decreases in free ATP levels and heat production, intracellular pH drops from 7.8 to 6.3 overnight. Use of isolated mitochondria to study transcriptional responses to anoxia offers several advantages: (1). the localized nature of transcript initiation, processing and degradation, all of which may be followed in organello; (2). the relatively simple cis- and trans-machinery involved and (3). the ability to provide relevant physiological treatments in vitro. In response to anoxic incubation of embryos in vivo for 4 h followed by anoxic mitochondrial isolation and anoxic transcription assay at pH 6.4, a significant decrease in overall UTP incorporation (77%) was seen after 30 min relative to normoxic, pH 7.9 controls. A less severe inhibition of transcription under anoxia (52%) was observed compared with controls when pH was raised to 7.9. Similarly, under normoxia, the incubation at low pH (6.4) reduced transcription by 59%. Ribonuclease protection assays showed that the contribution of in vitro initiation during the assay fell from 78% at pH 7.9 to approximately 32% at pH 6.4 under either normoxic or anoxic conditions. DNA footprinting of putative transcriptional promoters revealed proteins at regular intervals upstream of the 12S rRNA in the control region, which previously had been indirectly inferred to contain promoters for H-strand transcription. The area between 1230 and 12065 contains a sequence in the tRNA(leu) gene believed to bind the transcription termination factor mTERF or TERM, and we provide the first evidence that this sequence is protein-bound in A. franciscana. However, our hypothesis that initiation is reduced at low pH because of a change in DNA binding by mitochondrial transcription factors was not confirmed. We propose that regulation of initiation may be mediated by covalent modification or by protein-protein interactions not detected by footprinting.