Patricia Estes

Substrate specificity of the dsRNA unwinding/modifying activity.

Double‐stranded RNA (dsRNA) unwinding/modifying activity, which is present in a wide range of eukaryotic cells, has been previously shown to convert up to 50% of adenosine residues to inosines within intermolecular dsRNA. In the present study, we report that this activity also modifies, though slightly less efficiently, intramolecular double‐stranded regions of synthetic RNAs. Our results widen the range of the possible biological substrates for the activity since many stem and loop type RNA secondary structures (intramolecular dsRNA), present in eukaryotic as well as viral transcripts, can potentially serve as substrates. In addition, we have found that the dsRNA unwinding/modifying activity requires a double‐stranded region of at least 15–20 base pairs (bp) for substrate recognition. Furthermore, modification efficiency was found to be critically dependent on the length of the double‐stranded region; as the size decreased below 100 bp, it dropped precipitously. Our results suggest that efficient modification may occur only with relatively long (greater than 100 bp) dsRNA, perhaps because multiple copies of the enzyme must be bound.

Development of Morphological Diversity of Dendrites in Drosophila by the BTB-Zinc Finger Protein Abrupt

Morphological diversity of dendrites contributes to specialized functions of individual neurons. In the present study, we examined the molecular basis that generates distinct morphological classes of Drosophila dendritic arborization (da) neurons. da neurons are classified into classes I to IV in order of increasing territory size and/or branching complexity. We found that Abrupt (Ab), a BTB-zinc finger protein, is expressed selectively in class I cells. Misexpression of ab in neurons of other classes directed them to take the appearance of cells with smaller and/or less elaborated arbors. Loss of ab functions in class I neurons resulted in malformation of their typical comb-like arbor patterns and generation of supernumerary branch terminals. Together with the results of monitoring dendritic dynamics of ab-misexpressing cells or ab mutant ones, all of the data suggested that Ab endows characteristics of dendritic morphogenesis of the class I neurons.

Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns.

Stably transfected cell lines containing the normal human growth hormone (hGH-N) and human growth hormone-variant (hGH-V) genes have been established in order to study the expression of these two highly homologous genes. Each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse C127 cells. The transfected cells containing the hGH-N gene secrete two hGH proteins, 91% migrating at 22 kD and 9% migrating at 20 kD, the same relative proportions synthesized in vivo by the human pituitary. S1 nuclease analysis of mRNA from these cells confirms that 20 kD hGH is encoded by an alternatively spliced product of the primary hGH-N gene transcript in which the normal exon 3 splice-acceptor site is bypassed for a secondary site 15 codons within exon 3. Although the hGH-V gene is identical to the hGH-N gene for at least 15 nucleotides on either side of the normal and alternative exon 3 AG splice-acceptor sites, hGH-V synthesizes only a 22-kD protein. Reciprocal exchange of exon 3 and its flanking intron sequences between the hGH-N gene and the hGH-V gene, eliminates the synthesis of the 20-kD protein in both resultant chimeric genes. These results directly demonstrate that both the major 22-kD and the minor 20-kD forms of pituitary hGH are encoded by the alternative splicing products of a single hGH-N gene transcript. This alternative splicing is neither species nor tissue-specific and appears to be regulated by at least two separate regions remote from the AG splice-acceptor site.

Interpretation of X Chromosome Dose at Sex-lethal Requires Non-E-Box Sites for the Basic Helix-Loop-Helix Proteins SISB and Daughterless

ABSTRACT For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB(sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da)proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding atSxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter SxlPe . Surprisingly, most SISB-Da sites at SxlPe differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for SxlPe activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the SxlPe switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.

Identification of Motifs That Are Conserved in 12 Drosophila Species and Regulate Midline Glia vs . Neuron Expression

Functional complexity of the central nervous system (CNS) is reflected by the large number and diversity of genes expressed in its many different cell types. Understanding the control of gene expression within cells of the CNS will help reveal how various neurons and glia develop and function. Midline cells of Drosophila differentiate into glial cells and several types of neurons and also serve as a signaling center for surrounding tissues. Here, we examine regulation of the midline gene, wrapper, required for both neuron–glia interactions and viability of midline glia. We identify a region upstream of wrapper required for midline expression that is highly conserved (87%) between 12 Drosophila species. Site-directed mutagenesis identifies four motifs necessary for midline glial expression: (1) a Single-minded/Tango binding site, (2) a motif resembling a pointed binding site, (3) a motif resembling a Sox binding site, and (4) a novel motif. An additional highly conserved 27 bp are required to restrict expression to midline glia and exclude it from midline neurons. These results suggest short, highly conserved genomic sequences flanking Drosophila midline genes are indicative of functional regulatory regions and that small changes within these sequences can alter the expression pattern of a gene.

Expansion of Human and Murine Hematopoietic Stem and Progenitor Cells Ex Vivo without Genetic Modification Using MYC and Bcl-2 Fusion Proteins

The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.

Common motifs shared by conserved enhancers of Drosophila midline glial genes

Coding sequences are usually the most highly conserved sectors of DNA, but genomic regions controlling the expression pattern of certain genes can also be conserved across diverse species. In this study, we identify five enhancers capable of activating transcription in the midline glia of Drosophila melanogaster and each contains sequences conserved across at least 11 Drosophila species. In addition, the conserved sequences contain reiterated motifs for binding sites of the known midline transcriptional activators, Single-minded, Tango, Dichaete, and Pointed. To understand the molecular basis for the highly conserved genomic subregions within enhancers of the midline genes, we tested the ability of various motifs to affect midline expression, both individually and in combination, within synthetic reporter constructs. Multiple copies of the binding site for the midline regulators Single-minded and Tango can drive expression in midline cells; however, small changes to the sequences flanking this transcription factor binding site can inactivate expression in midline cells and activate expression in tracheal cells instead. For the midline genes described in this study, the highly conserved sequences appear to juxtapose positive and negative regulatory factors in a configuration that activates genes specifically in the midline glia, while maintaining them inactive in other tissues, including midline neurons and tracheal cells.

Specificity of the Receptor for the Major Sex Pheromone Component in Heliothis virescens

Abstract In a previous study, the Drosophila melanogaster OR67dGAL4;UAS system was used to functionally characterize the receptor for the major component of the sex pheromone in the tobacco budworm, Heliothis virescens Fabricius (Lepidoptera: Noctuidae), HvOR13. Electrophysiological and behavioral assays showed that transgenic flies expressing HvOR13 responded to (Z)-11-hexadecenal (Z11-16:Ald). However, tests were not performed to determine whether these flies would also respond to secondary components of the H. virescens sex pheromone. Thus, in this study the response spectrum of HvOR13 expressed in this system was examined by performing single cell recordings from odor receptor neuron in trichoid T1 sensilla on antennae of two Or67dGAL4 [1]; UAS-HvOR13 lines stimulated with Z11-16:Ald and six H. virescens secondary pheromone components. Fly courtship assays were also performed to examine the behavioral response of the Or67dGAL4[1]; UAS-HvOR13 flies to Z11-16:Ald and the secondary component Z9-14:Ald. Our combined electrophysiological and behavioral studies indicated high specificity and sensitivity of HvOR13 to Z11-16:Ald. Interestingly, a mutation leading to truncation in the HvOR13 C-terminal region affected but did not abolish pheromone receptor response to Z11-16:Ald. The findings are assessed in relationship to other HvOR13 heterologous expression studies, and the role of the C-terminal domain in receptor function is discussed. A third line expressing HvOR15 was also tested but did not respond to any of the seven pheromone components.

A comparison of midline and tracheal gene regulation during Drosophila development.

Within the Drosophila embryo, two related bHLH-PAS proteins, Single-minded and Trachealess, control development of the central nervous system midline and the trachea, respectively. These two proteins are bHLH-PAS transcription factors and independently form heterodimers with another bHLH-PAS protein, Tango. During early embryogenesis, expression of Single-minded is restricted to the midline and Trachealess to the trachea and salivary glands, whereas Tango is ubiquitously expressed. Both Single-minded/Tango and Trachealess/Tango heterodimers bind to the same DNA sequence, called the CNS midline element (CME) within cis-regulatory sequences of downstream target genes. While Single-minded/Tango and Trachealess/Tango activate some of the same genes in their respective tissues during embryogenesis, they also activate a number of different genes restricted to only certain tissues. The goal of this research is to understand how these two related heterodimers bind different enhancers to activate different genes, thereby regulating the development of functionally diverse tissues. Existing data indicates that Single-minded and Trachealess may bind to different co-factors restricted to various tissues, causing them to interact with the CME only within certain sequence contexts. This would lead to the activation of different target genes in different cell types. To understand how the context surrounding the CME is recognized by different bHLH-PAS heterodimers and their co-factors, we identified and analyzed novel enhancers that drive midline and/or tracheal expression and compared them to previously characterized enhancers. In addition, we tested expression of synthetic reporter genes containing the CME flanked by different sequences. Taken together, these experiments identify elements overrepresented within midline and tracheal enhancers and suggest that sequences immediately surrounding a CME help dictate whether a gene is expressed in the midline or trachea.

Mastermind mutations generate a unique constellation of midline cells within the Drosophila CNS.

Background The Notch pathway functions repeatedly during the development of the central nervous system in metazoan organisms to control cell fate and regulate cell proliferation and asymmetric cell divisions. Within the Drosophila midline cell lineage, which bisects the two symmetrical halves of the central nervous system, Notch is required for initial cell specification and subsequent differentiation of many midline lineages. Methodology/Principal Findings Here, we provide the first description of the role of the Notch co-factor, mastermind, in the central nervous system midline of Drosophila. Overall, zygotic mastermind mutations cause an increase in midline cell number and decrease in midline cell diversity. Compared to mutations in other components of the Notch signaling pathway, such as Notch itself and Delta, zygotic mutations in mastermind cause the production of a unique constellation of midline cell types. The major difference is that midline glia form normally in zygotic mastermind mutants, but not in Notch and Delta mutants. Moreover, during late embryogenesis, extra anterior midline glia survive in zygotic mastermind mutants compared to wild type embryos. Conclusions/Significance This is an example of a mutation in a signaling pathway cofactor producing a distinct central nervous system phenotype compared to mutations in major components of the pathway.