Lisa A. Meadows

Frequency and Voltage Dependence of the Dielectrophoretic Trapping of Short Lengths of DNA and dCTP in a Nanopipette

The study of the properties of DNA under high electric fields is of both fundamental and practical interest. We have exploited the high electric fields produced locally in the tip of a nanopipette to probe the motion of double- and single-stranded 40-mer DNA, a 1-kb single-stranded DNA, and a single-nucleotide triphosphate (dCTP) just inside and outside the pipette tip at different frequencies and amplitudes of applied voltages. We used dual laser excitation and dual color detection to simultaneously follow two fluorophore-labeled DNA sequences with millisecond time resolution, significantly faster than studies to date. A strong trapping effect was observed during the negative half cycle for all DNA samples and also the dCTP. This effect was maximum below 1 Hz and decreased with higher frequency. We assign this trapping to strong dielectrophoresis due to the high electric field and electric field gradient in the pipette tip. Dielectrophoresis in electrodeless tapered nanostructures has potential applications for controlled mixing and manipulation of short lengths of DNA and other biomolecules, opening new possibilities in miniaturized biological analysis.

Stability and Dynamics of Polycomb Target Sites in Drosophila Development

Polycomb-group (PcG) and Trithorax-group proteins together form a maintenance machinery that is responsible for stable heritable states of gene activity. While the best-studied target genes are the Hox genes of the Antennapedia and Bithorax complexes, a large number of key developmental genes are also Polycomb (Pc) targets, indicating a widespread role for this maintenance machinery in cell fate determination. We have studied the linkage between the binding of PcG proteins and the developmental regulation of gene expression using whole-genome mapping to identify sites bound by the PcG proteins, Pc and Pleiohomeotic (Pho), in the Drosophila embryo and in a more restricted tissue, the imaginal discs of the third thoracic segment. Our data provide support for the idea that Pho is a general component of the maintenance machinery, since the majority of Pc targets are also associated with Pho binding. We find, in general, considerable developmental stability of Pc and Pho binding at target genes and observe that Pc/Pho binding can be associated with both expressed and inactive genes. In particular, at the Hox complexes, both active and inactive genes have significant Pc and Pho binding. However, in comparison to inactive genes, the active Hox genes show reduced and altered binding profiles. During development, Pc target genes are not simply constantly associated with Pc/Pho binding, and we identify sets of genes with clear differential binding between embryo and imaginal disc. Using existing datasets, we show that for specific fate-determining genes of the haemocyte lineage, the active state is characterised by lack of Pc binding. Overall, our analysis suggests a dynamic relationship between Pc/Pho binding and gene transcription. Pc/Pho binding does not preclude transcription, but levels of Pc/Pho binding change during development, and loss of Pc/Pho binding can be associated with both stable gene activity and inactivity.

Genomic mapping of Suppressor of Hairy-wing binding sites in Drosophila.

BackgroundInsulator elements are proposed to play a key role in the organization of the regulatory architecture of the genome. In Drosophila, one of the best studied is the gypsy retrotransposon insulator, which is bound by the Suppressor of Hairy-wing (Su [Hw]) transcriptional regulator. Immunolocalization studies suggest that there are several hundred Su(Hw) sites in the genome, but few of these endogenous Su(Hw) binding sites have been identified.ResultsWe used chromatin immunopurification with genomic microarray analysis to identify in vivo Su(Hw) binding sites across the 3 megabase Adh region. We find 60 sites, and these enabled the construction of a robust new Su(Hw) binding site consensus. In contrast to the gypsy insulator, which contains tightly clustered Su(Hw) binding sites, endogenous sites generally occur as isolated sites. These endogenous sites have three key features. In contrast to most analyses of DNA-binding protein specificity, we find that strong matches to the binding consensus are good predictors of binding site occupancy. Examination of occupancy in different tissues and developmental stages reveals that most Su(Hw) sites, if not all, are constitutively occupied, and these isolated Su(Hw) sites are generally highly conserved. Analysis of transcript levels in su(Hw) mutants indicate widespread and general changes in gene expression. Importantly, the vast majority of genes with altered expression are not associated with clustering of Su(Hw) binding sites, emphasizing the functional relevance of isolated sites.ConclusionTaken together, our in vivo binding and gene expression data support a role for the Su(Hw) protein in maintaining a constant genomic architecture.

Variable sexually dimorphic gene expression in laboratory strains of Drosophila melanogaster

BackgroundWild-type laboratory strains of model organisms are typically kept in isolation for many years, with the action of genetic drift and selection on mutational variation causing lineages to diverge with time. Natural populations from which such strains are established, show that gender-specific interactions in particular drive many aspects of sequence level and transcriptional level variation. Here, our goal was to identify genes that display transcriptional variation between laboratory strains of Drosophila melanogaster, and to explore evidence of gender-biased interactions underlying that variability.ResultsTranscriptional variation among the laboratory genotypes studied occurs more frequently in males than in females. Qualitative differences are also apparent to suggest that genes within particular functional classes disproportionately display variation in gene expression. Our analysis indicates that genes with reproductive functions are most often divergent between genotypes in both sexes, however a large proportion of female variation can also be attributed to genes without expression in the ovaries.ConclusionThe present study clearly shows that transcriptional variation between common laboratory strains of Drosophila can differ dramatically due to sexual dimorphism. Much of this variation reflects sex-specific challenges associated with divergent physiological trade-offs, morphology and regulatory pathways operating within males and females.

Conserved properties of Drosophila and human spermatozoal mRNA repertoires

It is now well established that mature mammalian spermatozoa carry a population of mRNA molecules, at least some of which are transferred to the oocyte at fertilization, however, their function remains largely unclear. To shed light on the evolutionary conservation of this feature of sperm biology, we analysed highly purified populations of mature sperm from the fruitfly, Drosophila melanogaster. As with mammalian sperm, we found a consistently enriched population of mRNA molecules that are unlikely to be derived from contaminating somatic cells or immature sperm. Using tagged transcripts for three of the spermatozoal mRNAs, we demonstrate that they are transferred to the oocyte at fertilization and can be detected before, and at least until, the onset of zygotic gene expression. We find a remarkable conservation in the functional annotations associated with fly and human spermatozoal mRNAs, in particular, a highly significant enrichment for transcripts encoding ribosomal proteins (RPs). The substantial functional coherence of spermatozoal transcripts in humans and the fly opens the possibility of using the power of Drosophila genetics to address the function of this enigmatic class of molecules in sperm and in the oocyte following fertilization.

Neighbourhood Continuity Is Not Required for Correct Testis Gene Expression in Drosophila

Disrupting the linear organization of testis gene expression neighborhoods in the Drosophila genome does not affect gene expression, suggesting that neighborhood organization is not primarily driven by gene expression requirements.

The impact of quantitative optimization of hybridization conditions on gene expression analysis

BackgroundWith the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols.ResultsAs shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of up to 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays.For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We can ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirmed an unbiased determination of generally optimal experimental conditions.ConclusionsWell calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive pro filing of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks.

Targets of homeotic gene regulation in Drosophila

Summary We have used a chromatin immunopurification approach to identify target genes regulated by the homeotic gene Ultrabithorax. A monoclonal antibody against the Ultrabithorax gene product is used to immunopurify in vivo Ultrabithorax protein binding sites in embryonic chromatin. The procedure gives an enrichment of sequences with matches to a consensus homeodomain binding site. In one case we have shown that an immunopurified sequence lies within a 4 kb fragment that acts in vivo as a homeotic response element. We anticipate that this approach will enable us to identify further targets, allowing the analysis of their regulation and function. The chromatin immunopurification strategy may be of general application for the identification of direct in vivo targets of DNA-binding proteins.

SimArray: a user-friendly and user-configurable microarray design tool.

BackgroundMicroarrays were first developed to assess gene expression but are now also used to map protein-binding sites and to assess allelic variation between individuals. Regardless of the intended application, efficient production and appropriate array design are key determinants of experimental success. Inefficient production can make larger-scale studies prohibitively expensive, whereas poor array design makes normalisation and data analysis problematic.ResultsWe have developed a user-friendly tool, SimArray, which generates a randomised spot layout, computes a maximum meta-grid area, and estimates the print time, in response to user-specified design decisions. Selected parameters include: the number of probes to be printed; the microtitre plate format; the printing pin configuration, and the achievable spot density. SimArray is compatible with all current robotic spotters that employ 96-, 384- or 1536-well microtitre plates, and can be configured to reflect most production environments. Print time and maximum meta-grid area estimates facilitate evaluation of each array design for its suitability. Randomisation of the spot layout facilitates correction of systematic biases by normalisation.ConclusionSimArray is intended to help both established researchers and those new to the microarray field to develop microarray designs with randomised spot layouts that are compatible with their specific production environment. SimArray is an open-source program and is available from http://www.flychip.org.uk/SimArray/.

Sugar and spice or slugs and snails

Over the past 500 million years or so, metazoans have evolved a bewildering diversity of genetic mechanisms for generating that most interesting of phenotypes — the difference between girls and boys. Despite intense effort, a conserved sex-determination effector gene has proven elusive. Until recently, the evidence indicated that completely different molecular mechanisms and proteins regulate sexual dimorphism in different phyla; sex-specific RNA splicing in flies, a signal transduction cascade in worms and a male-determining DNA-binding protein in mammals. Could it be that the quest is now over and a single unifying gene is at the bottom of it all?Recent evidence from studies on a class of transcription factors known as DM-domain proteins has implicated a common effector in the regulation of male-specific characteristics in flies, nematodes, reptiles, birds and mammals. Mutations in the Drosophila doublesex (dsx) and Caenorhabditis elegans mab-3 genes lead to defects in a variety of male-specific processes. As well as being functionally interchangeable, the genes regulate similar male-specific biological processes, such as repressing yolk-protein synthesis and eliciting the development of a sex-specific nervous system. Furthermore, a vertebrate DM-domain gene, DMRT1, which is associated with a small interval of chromosome 9 required for human testis development, exhibits male-specific expression in the gonads of chickens and alligators. This indicates that diverged sex-determining mechanisms might converge at a common effector gene that functions to promote male development.Raymond et al. 1xDmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Raymond, C.S. et al. Genes Dev. 2000; 14: 2587–2595Crossref | PubMed | Scopus (374)See all References1 have examined this possibility by generating mice with mutations in Dmrt1. They report that, whereas female development is normal in mouse Dmrt1 knockouts, the differentiation of the testis is obviously not. Testis differentiation is the key event in mammalian sex determination, in particular the formation of a somatic support-cell lineage known as Sertoli cells. It is thought that the mammalian sex-determining gene Sry acts to promote the differentiation of Sertoli cells, and that these influence the subsequent events in testis differentiation. In Dmrt1-mutant males, the testes appear normal at birth, but by the onset of germ-cell meiosis a few days later, abnormalities are apparent. After two weeks, the mutant testes are filled with immature Sertoli cells, a phenotype resembling that observed in human chromosome 9-deficient males.Thus, it appears that a DM-domain gene functions as a downstream effector in vertebrate sexual differentiation in a way analogous to that observed in invertebrates. Interestingly, the chromosome 9 interval associated with DMRT1 in humans contains at least two other DM-domain genes. The situation in the mouse is not clear, but it could be that multiple DM-domain genes have evolved in mammals to ensure correct male differentiation. Clearly, a functional analysis of DMRT1 in other organisms will go a long way to confirm whether the evolution of sexual dimorphism has, after all, been conserved.