Metal-Free CVD Graphene Synthesis on 200 mm Ge/Si(001) Substrates.

Good quality, complementary-metal-oxide-semiconductor (CMOS) technology compatible, 200 mm graphene was obtained on Ge(001)/Si(001) wafers in this work. Chemical vapor depositions were carried out at the deposition temperatures of 885 °C using CH4 as carbon source on epitaxial Ge(100) layers, which were grown on Si(100), prior to the graphene synthesis. Graphene layer with the 2D/G ratio ∼3 and low D mode (i.e., low concentration of defects) was measured over the entire 200 mm wafer by Raman spectroscopy. A typical full-width-at-half-maximum value of 39 cm-1 was extracted for the 2D mode, further indicating that graphene of good structural quality was produced. The study also revealed that the lack of interfacial oxide correlates with superior properties of graphene. In order to evaluate electrical properties of graphene, its 2 × 2 cm2 pieces were transferred onto SiO2/Si substrates from Ge/Si wafers. The extracted sheet resistance and mobility values of transferred graphene layers were ∼1500 ± 100 Ω/sq and µ ≈ 400 ± 20 cm2/V s, respectively. The transferred graphene was free of metallic contaminations or mechanical damage. On the basis of results of DFT calculations, we attribute the high structural quality of graphene grown by CVD on Ge to hydrogen-induced reduction of nucleation probability, explain the appearance of graphene-induced facets on Ge(001) as a kinetic effect caused by surface step pinning at linear graphene nuclei, and clarify the orientation of graphene domains on Ge(001) as resulting from good lattice matching between Ge(001) and graphene nucleated on such nuclei.

Tantalus, a novel ASX-interacting protein with tissue-specific functions.

The Drosophila trithorax- and Polycomb-group (trxG and PcG) proteins maintain activated and repressed transcriptional states at specific target gene loci. The Additional sex combs (Asx) gene is of particular interest as it appears to function in both protein complexes and yet its effects on target genes are more restricted. A novel protein, Tantalus (TAN), was identified in a yeast two-hybrid screen for ASX-interacting proteins that might confer tissue-specific ASX functions. TAN contains consensus nuclear localization sites and binds DNA in vitro. However, its subcellular localization varies in a tissue-specific fashion. In salivary glands, TAN is predominantly nuclear and associates with 66 euchromatic sites on polytene chromosomes, more than half of which overlap with ASX. These loci do not include the homeotic genes of the ANT and BX complexes bound by other PcG and trxG proteins. Rather, tan mutant defects are restricted to sensory organs. We show that one of these defects, shared by Asx, is genetically enhanced by Asx. Taken together, the data suggest that TAN is a tissue-specific cofactor for ASX, and that its activity may be partially controlled by subcellular trafficking.

Apical localization of wingless transcripts is required for wingless signaling.

Many developing and adult tissues are comprised of polarized epithelia. Proteins that are asymmetrically distributed in these cells are thought to be localized by protein trafficking. Here we show that the distribution and function of the signaling protein Wingless is predetermined by the subcellular localization of its mRNA. High-resolution in situ hybridization reveals apical transcript localization in the majority of tissues examined. This localization is mediated by two independently acting elements in the 3' UTR. Replacement of these elements with non- or basolaterally localizing elements yields proteins with altered intracellular and extracellular distributions and reduced signaling activities. This novel aspect of the wingless signaling pathway is conserved and may prove to be a mechanism used commonly for establishing epithelial cell polarity.

Establishment and maintenance of parasegmental compartments.

Embryos of higher metazoans are divided into repeating units early in development. In Drosophila, the earliest segmental units to form are the parasegments. Parasegments are initially defined by alternating stripes of expression of the fushi-tarazu and even-skipped genes. How fushi-tarazu and even-skipped define the parasegment boundaries, and how parasegments are lost when fushi-tarazu or even-skipped fail to function correctly, have never been fully or properly explained. Here we show that parasegment widths are defined early by the relative levels of fushi-tarazu and even-skipped at stripe junctions. Changing these levels results in alternating wide and narrow parasegments. When shifted by 30% or more, the enlarged parasegments remain enlarged and the reduced parasegments are lost. Loss of the reduced parasegments occurs in three steps; delamination of cells from the epithelial layer, apoptosis of the delaminated cells and finally apoptosis of inappropriate cells remaining at the surface. The establishment and maintenance of vertebrate metameres may be governed by similar processes and properties.

FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs.

To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We also show that FTZ contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins.

Mechanisms regulating target gene selection by the homeodomain-containing protein Fushi tarazu.

Homeodomain proteins are DNA-binding transcription factors that control major developmental patterning events. Although DNA binding is mediated by the homeodomain, interactions with other transcription factors play an unusually important role in the selection and regulation of target genes. A major question in the field is whether these cofactor interactions select target genes by modulating DNA binding site specificity (selective binding model), transcriptional activity (activity regulation model) or both. A related issue is whether the number of target genes bound and regulated is a small or large percentage of genes in the genome. In this study, we have addressed these issues using a chimeric protein that contains the strong activation domain of the viral VP16 protein fused to the Drosophila homeodomain-containing protein Fushi tarazu (Ftz). We find that genes previously thought not to be direct targets of Ftz remain unaffected by FtzVP16. Addition of the VP16 activation domain to Ftz does, however, allow it to regulate previously identified target genes at times and in regions that Ftz alone cannot. It also changes Ftz into an activator of two genes that it normally represses. Taken together, the results suggest that Ftz binds and regulates a relatively limited number of target genes, and that cofactors affect target gene specificity primarily by controlling binding site selection. Activity regulation then fine-tunes the temporal and spatial domains of promoter responses, the magnitude of these responses, and whether they are positive or negative.

Tissue-specific requirements for a phosphorylation site in the Fushi tarazu homeodomain.

The homeodomain protein Fushi tarazu (Ftz) is required for several embryonic patterning processes including segmentation and neurogenesis. During the stages that these processes are regulated the protein is differentially phosphorylated, suggesting that phosphorylation plays a role in helping the protein to regulate different functions in different tissues. We showed in a recent study that one of the Ftz phosphorylation sites, a protein kinase A-type site in the N-terminal arm of the homeodomain, is required for normal Ftz-dependent segmentation. Here we test whether phosphorylation of this site (Thr-263) is also required in the developing central nervous system (CNS). A well-established role for Ftz in the CNS is for the differentiation of neurons referred to as RP2 neurons. Absence of Ftz expression in these cells causes a failure of certain target genes to be expressed and subsequent defects in RP2 differentiation. In contrast to its effect on segmentation, we find that mutation of Thr-263 to Ala (or Asp) has no effect on these CNS functions. This suggests that the phosphorylation state of this site is irrelevant for Ftz function in the CNS, and that there are tissue-specific differences in the requirements for Ftz phosphorylation.

Kinetic analysis of segmentation gene interactions in Drosophila embryos.

A major challenge for developmental biologists in coming years will be to place the vast number of newly identified genes into precisely ordered genetic and molecular pathways. This will require efficient methods to determine which genes interact directly and indirectly. One of the most comprehensive pathways currently under study is the genetic hierarchy that controls Drosophila segmentation. Yet, many of the potential interactions within this pathway remain untested or unverified. Here, we look at one of the best-characterized components of this pathway, the homeodomain-containing transcription factor Fushi tarazu (Ftz), and analyze the response kinetics of known and putative target genes. This is achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, we find that both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (<10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes are analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy.

Molecular interactions between Vestigial and Scalloped promote wing formation in Drosophila.

Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development. We show that Sd is required for Vg function and that altering their relative cellular levels inhibits wing formation. In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. Our observations indicate that Vg and Sd function coordinately to control the expression of genes required for wing development, which implies that Vg is a tissue-specific transcriptional intermediary factor of Sd.

Dynamic changes in the functions of Odd-skipped during early Drosophila embryogenesis.

Although many of the genes that pattern the segmented body plan of the Drosophila embryo are known, there remains much to learn in terms of how these genes and their products interact with one another. Like many of these gene products, the protein encoded by the pair-rule gene odd-skipped (Odd) is a DNA-binding transcription factor. Genetic experiments have suggested several candidate target genes for Odd, all of which appear to be negatively regulated. Here we use pulses of ectopic Odd expression to test the response of these and other segmentation genes. The results are complex, indicating that Odd is capable of repressing some genes wherever and whenever Odd is expressed, while the ability to repress others is temporally or spatially restricted. Moreover, one target gene, fushi tarazu, is both repressed and activated by Odd, the outcome depending upon the stage of development. These results indicate that the activity of Odd is highly dependent upon the presence of cofactors and/or overriding inhibitors. Based on these results, and the segmental phenotypes generated by ectopic Odd, we suggest a number of new roles for Odd in the patterning of embryonic segments. These include gap-, pair-rule- and segment polarity-type functions.

A phosphorylation site in the ftz homeodomain is required for activity.

The Drosophila homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. Here we show that one of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala resulted in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescued the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. We also demonstrate that the T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein.

DNA gyrase inhibitory and antimicrobial activities of some diphenic acid monohydroxamides.

The synthesis and inhibitory activity against DNA gyrase of a series of diphenic acid monohydroxamides 4a-f are described. A protocol of two biological assays showed conclusively that inhibition occurs specifically at the DNA-DNA gyrase complex and is not attributable to nonspecific inhibition. In the enzyme assays, 4c was potent as the prototypical quinolone, nalidixic acid (1), with an IC50 value of 58.3 micrograms/mL compared to 52 micrograms/mL for 1. MIC activity against bacterial strains showed a systematic drop for all compounds relative to 1. For compounds 4c-e, the addition of PMBN produced dramatic increases in MIC activity indicating that activity is likely to be related to membrane transport. Molecular modeling of 4a indicates that the diphenic acid monohydroxamides can bind to the DNA-DNA gyrase complex in a similar fashion as that hypothesized for the quinolone series according to the hypothesis suggested by Shen et al. but may not self-associate by pi-pi stacking. In contrast to the quinolone series, as the diphenic acid monohydroxamides are shown by molecular mechanics minimizations to be nonplanar, they may present novel approaches for chemotherapeutic intervention with a potential for decreased side effects.

The nuclear receptor homologue Ftz-F1 and the homeodomain protein Ftz are mutually dependent cofactors.

Nuclear hormone receptors and homeodomain proteins are two classes of transcription factor that regulate major developmental processes. Both depend on interactions with other proteins for specificity and activity. The Drosophila gene fushi tarazu (ftz), which encodes a homeodomain protein (Ftz), is required zygotically for the formation of alternate segments in the developing embryo. Here we show that the orphan nuclear receptor alphaFtz-F1 (ref. 3), which is deposited in the egg during oogenesis, is an obligatory cofactor for Ftz. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. This interaction suggests that other nuclear receptor/homeodomain protein interactions maybe important and common in developing organisms.

Patterning of the Drosophila embryo by a homeodomain-deleted Ftz polypeptide.

Homeodomain proteins regulate diverse developmental processes in a wide range of organisms, yet bind in vitro to DNA sequences that are remarkably similar. This has raised the fundamental question of how target gene specificity is achieved in vivo. The Drosophila fushi tarazu protein (Ftz) contains a homeodomain and is required for the formation of alternate segments. We have shown previously that a homeodomain-deleted Ftz polypeptide (Ftz delta HD), incapable of binding DNA in vitro, could regulate endogenous ftz gene expression. Here we test Ftz delta HD activities in a ftz mutant background and find that, surprisingly, Ftz delta HD can directly regulate ftz-dependent segmentation, suggesting that it can control target gene expression through interactions with other proteins. A likely candidate is the pair-rule protein Paired (Prd). Ftz delta HD bound directly to Prd in vitro and required Prd to repress wingless in vivo. These results emphasize the pivotal importance of protein-protein interactions in homeodomain protein function.

A comparison of active site binding of 4-quinolones and novel flavone gyrase inhibitors to DNA gyrase.

The activity of 4-quinolone antibacterials at the enzyme target level is based on the well known and reported observations that 4-quinolone antibacterials target the Gyr A subunit of the DNA gyrase holoenzyme, inhibiting supercoiling while facilitating the "cleavable complex". Such inhibition can be observed by running the in vitro DNA gyrase supercoiling inhibition assay or the "cleavable complex" DNA gyrase assay. Although potency of the gyrase inhibitor is dependent on many factors including permeability and pharmacokinetics, the inherent potency of a gyrase inhibitor lies in its activity against the target enzyme. We have examined the binding activity of novel flavones [Bioorganic & Med. Chem. Letters 3:225-230, 1993] to Escherichia coli DNA gyrase and have found differences in binding consistent with inhibition of DNA gyrase supercoiling and ability to facilitate the cleavable complex, but of different rank order. [3H]norfloxacin was used in vitro competition studies with test compounds, pBR322 and E. coli DNA gyrase. Binding affinity results indicate the rank order of greatest to weakest binding (ability to compete with [3H]norfloxacin) of test compounds: Levofloxacin = ciprofloxacin > ofloxacin > norfloxacin > flavone compounds (including ellagic acid, quercetin, and compounds 5a through 5n [Bioorganic & Med. Chem. Letters 3:225-230, 1993]). Such differences in binding ability of the 4-quinolones and flavones to the ternary complex of DNA.DNA gyrase.drug, as compared to the catalytic inhibition and "cleavable complex" data, suggests a more complex binding of flavones than the previously hypothesized models for 4-quinolone binding.

Testing potential gyrase inhibitors of bacterial DNA gyrase: a comparison of the supercoiling inhibition assay and "cleavable complex" assay.

Inhibitory activity of test compounds against Escherichia coli DNA gyrase in a "cleavable complex" assay, readily observed in vitro at the enzyme level by the artificial addition of a denaturing agent, is found to be an excellent indicator of 4-quinolone inhibition of DNA gyrase, and as accurate a predictor of target enzyme inhibitory activity as the measurement of the inhibition of DNA gyrase supercoiling. This study was designed to examine the specificity of DNA gyrase inhibitors of various chemical classes in these two DNA gyrase assays, and define the use of these two assays in understanding the nature of inhibition by experimental compounds. Supercoiling inhibition was detected by determination of the 50% inhibition level, and cleavable complex inhibition measured by the determination of the drug concentration at which 50% of the maximal (of control) formation of linear, cleaved DNA was obtained. Results indicate that these two assays can serve several different functions in microbiological research, among them: (1) quantitation of enzyme inhibitory activity at the target level; and (2) distinguishing between nonspecific inhibition or artifactual inhibition of DNA gyrase and true, mechanism-based inhibition of the catalytic activity of DNA gyrase.

Control of segmental asymmetry in Drosophila embryos.

During Drosophila development, an important aspect of body patterning is the division of the embryo into repeating morphological units referred to as parasegments. The parasegmental domains are first defined at the blastoderm stage by alternating stripes of transcripts encoded by the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) and later by stripes encoded by the segment polarity genes engrailed (en) and wingless. Here, we show that the runt gene (run) is required to generate asymmetries within these parasegmental domains. Using a heat-shock-inducible run transgene, we found that ectopic run expression leads to rapid repression of eve stripes and a somewhat delayed expansion of ftz stripes. Unexpectedly, we also found that ectopic run was a rapid and potent repressor of odd-numbered en stripes. Two remarkably different segmental phenotypes were generated as a consequence of these effects. In solving the mechanisms underlying these phenotypes, we discovered that the positioning of en stripes is largely determined by the actions of negative regulators. Our data indicate that run is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of run and odd by the product of the eve gene. The spatial restriction of gene expression via negative and double negative pathways such as these is likely to be a common theme during development.