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The genes of the trithorax group (trxG) inDrosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes. The precise role of each of the diverse trxG members and the functional relationships among them are not well understood. Here, we report on the isolation of the trxG gene moira(mor) and its molecular characterization. morencodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3. mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM. MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes. These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.
Two classes of genes maintain regulatory decisions made during early Drosophiladevelopment by the localized expression of segmentation gene products. These are the Polycomb group (PcG) and thetrithorax group (trxG) genes, which sustain, respectively, the repressed or active state of homeotic gene expression (30, 42). The trxG proteins are thought to act at many different levels of gene regulation to maintain continued and efficient expression of homeotic and other genes. While genetic tests indicate that members of this group are functionally related (see, for example, references 41 and 49), with the exception of Brahma and Snf5-related 1 (SNR1) (14), there has been no evidence that these proteins are components of the same multimeric complexes. Thus, the exact functional relationships among most members of this gene class are not yet understood.
Brahma (BRM) is a trxG protein product that is believed to increase target gene accessibility by overcoming the repressive effects of nucleosomal histones (9, 45). BRM is highly related to theSaccharomyces cerevisiae protein SNF2 (SWI2) (44), which is part of an 11-subunit, 2-MDa global regulatory complex that assists a large number of DNA-binding proteins to activate transcription of their target genes by facilitating their binding to nucleosomal sites (31, 50). Another potential fruit fly homolog of a yeast SWI-SNF component that may be involved in the regulation of homeotic gene expression is SNR1, which coisolates with BRM in a large protein complex (14). Although it was not, like most of the trxG genes, isolated in screens that were based on suppression of mutations in Polycomb, Snr1does undergo genetic interactions with classic trxG genes (14).
To unravel the functional relationships among the different trxG members and to understand how the gene products exert regulatory control over other genes, it is necessary to obtain molecular information about those members that have not yet been cloned. One such gene is moira (mor), which was isolated in three independent screens for loci that undergo dosage-dependent interactions with Polycomb or ectopically expressedAntennapedia (29) and which exhibits many of the genetic and phenotypic characteristics of brm (6, 15, 16, 29, 44).
Here we demonstrate that mor encodes a Drosophilahomolog of the S. cerevisiae SWI-SNF gene SWI3. The MOR protein sequence closely resembles those of the human SWI3-related proteins, BAF170 and BAF155 (53, 54). In accordance with its known function as a regulator of homeotic gene activity, mor is widely expressed during development and in many embryonic tissues in a spatiotemporal pattern that overlaps that of brm and Snr1. MOR is capable of forming homooligomers; optimal binding of MOR to itself is likely to require its leucine zipper motif. MOR can also bind to BRM, with which it is associated in embryonic nuclear extracts; this interaction may be mediated by the SANT domain of MOR, which is common to several proteins involved in basal or activated transcription and whose function is not known (1, 54).
Identification of MOR as an additional component of theDrosophila SWI-SNF complex provides a physical and biochemical explanation for the known functional relationship between two strong and well-characterized trxG proteins, MOR and BRM. This finding provides a conceptual framework in which to continue to analyze the role of SWI-SNF proteins in regulation of the homeotic and other developmentally regulated genes in eukaryotic organisms.
Fruit fly culture and stocks. Drosophila melanogasterwas cultured at room temperature on standard cornmeal-yeast extract-dextrose medium or at 18°C on Instant Medium (Carolina Biological). Except for those generated in the course of this work, the mutations and transposons used are described in the online database FlyBase (20). mor region stocks and deficiencies were obtained from J. Kennison. All mor alleles were rebalanced over a TM3-ftz-lacZ balancer.
Isolation of new alleles of mor. The originalP{lacW} insertion mutagenesis was carried out with an attached-X ammunition chromosome that carries four copies ofP{lacW} on each arm: C(1)RM, y1? P{lacW}5-45fD w 1? P{lacW}4-5fP P{lacW}3-52dP{lacW}3-76a (24). Females carrying the attached-X ammunition chromosome and P{Δ2-3}99B were crossed with w; Sb sbd-2 Ubx bxd-1 males. F1 males with the mini-white eye color were crossed with w;Sb sbd-2 Ubx bxd-1 females. Whenever possible, homozygous insertion lines were established. F2 or F3 males were examined for changes in abdominal pigmentation or sternite bristle patterns. The insertion later designated P{lacW}89B mapped very close toSb on the basis of segregation with the originalSb sbd-2 mutation. Mobilization of this element was accomplished by generating dysgenic males, crossing withw − females, and recoveringw − Sb + animals. Of 47w − chromosomes recovered, 2 were lethal overDf(3R)sbd 105.
Cloning of the mor region and molecular analysis of new excision alleles of mor. The P{lacW}element allows cloning of adjacent genomic sequences by plasmid rescue (56). One isolate, pCK5.128A, obtained by EcoRI digestion of flies bearing P{lacW}89B, was labeled (random priming kit; Bethesda Research Laboratories) and used to screen a Sau3A partial genomic library of Canton-S DNA generated by using a Lambda-FIX kit (Stratagene) and high-molecular-weight genomic DNA (prepared as described in reference 34). Three independent genomic λ clones were recognized and characterized by restriction mapping and Southern analysis. Smaller genomic fragments were subcloned into pBluescript II SK (Stratagene).
The newly obtained excision mutants, mor 9 andmor 10, were subjected to Southern analysis withHindIII or EcoRI as described previously (12). As controls, the progenitor P{lacW}89Bchromosome and a line thought to be a precise excision thereof were also analyzed. Consistent evidence of changes in both lines was obtained by using the λ probe CK128γ (Fig.1A). When the original blot was reprobed with pP{CaSpeR} and pBluescript, there was no evidence that any portion of the P{lacW} element remained in eithermor 9 or mor 10.
Isolation of cDNAs and computer analyses.To obtain the cDNA corresponding to the 4.5-kb transcript, an early embryonic cDNA library (8) was screened. One positive clone was recovered, and the cDNA insert was subcloned into pBluescript II SK. DNA, prepared by using a Wizard Miniprep kit (Promega), was sequenced at the sequencing facility of the Life Sciences Institute, Hebrew University, Jerusalem, Israel. Synthetic primers were made to allow complete sequencing of both strands. To obtain additional cDNA clones, we screened a random-primed embryonic cDNA library (27) with 5′ sequences of the one positive clone and also carried out PCR amplification (with PWO [Boehringer]) on the same library with an oligonucleotide based on the sequence of the noncoding strand of Swi3D-2(5′ TTCAAAGCCCTGCAGCGTC 3′) and the λgt11 reverse primer. Sequences were analyzed by using the National Center for Supercomputer Applications electronic mail server and the Fasta (37), Blast (2), Gap (35), and Pileup (17) programs.
RNA preparation, Northern blotting, and determination of the direction of transcription.Developmental Northern blotting of total nucleic acids was performed by grinding frozen samples in a 7 M urea buffer, subjecting them to phenol-chloroform extractions and ethanol precipitation, and then applying approximately 12 μg of nucleic acid from each time point to a formaldehyde-morpholinepropanesulfonic acid (MOPS) denaturing gel (12).
To determine the direction of transcription of the 4.5-kb transcript, single-stranded RNA probes were generated from subclones of the 1.3-kbEcoRI fragment to the left of the P{lacW}89Binsertion, placed in both orientations into pBluescript II SK. These constructs were used to probe the total nucleic acid blots.
Southern blotting of DNA derived from fruit flies mutant inmor, and single-embryo PCR.Genomic DNA was isolated by homogenizing 200 flies in 1.6 ml of homogenization buffer (30 mM Tris-Cl [pH 9.0], 10 mM EDTA, 100 mM NaCl, 70 g of sucrose/liter) and 0.4 ml of lysis buffer (0.5 M Tris-Cl [pH 9.0], 0.25 M EDTA, 2.5% sodium dodecyl sulfate [SDS]), incubating at 65°C for 30 min, and, after the addition of 300 μl of 8 M potassium acetate, incubating for an additional hour at 0°C. Following centrifugation, 10 μg of ethanol-precipitated DNA was used per lane. Southern blots were prehybridized at 65°C in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 1% N -lauroylsarcosine (Sigma) and 0.5 mg of herring sperm DNA (Sigma)/ml and, following addition of the probe, hybridized overnight. The washes reached a stringency of 0.1× SSC–0.5% N -lauroylsarcosine. The Swi3D probe, encompassing 3.6 kb of Swi3D cDNA (excluding the 5′-most 180 bp ofSwi3D-1), was labeled by using a random primer labeling kit (Biological Industries, Beit HaEmek, Israel).
For PCR analysis of homozygous embryos, 3- to 12-h embryos laid by flies carrying mor alleles over a TM3-ftz-lacZbalancer were stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) (21). DNA was amplified by using a RedHot Taqpolymerase (Advanced Biotechnologies), and the amplified band was isolated for sequencing by using a QIAquick gel extraction kit (Qiagen).
P element transformation and rescue of the morphenotype.A genomic SalI fragment approximately 11.8 kb in length (Fig. 1E) was cloned into the XhoI site of the pP{CaSpeR-4} transformation vector (20). This fragment includes approximately 3 kb of flanking sequence at both the 5′ and 3′ ends of the 4.5-kb transcript, as well as the 5′ portion ofHel89B. Isolates with the fragment inserted in both orientations were recovered and designated pP{11.8A} and pP{11.8B}. The 4.5-kb mRNA is transcribed in the opposite direction relative to the CaSpeR mini-white gene in the pP{11.8A} construct.
y1 w1118 embryos were coinjected with pP{11.8} and pπ25.7wc in accordance with standard techniques (5, 43). Emerging adults were crossed individually to y1 w1118 flies, and transformants were identified. Two independent lines were recovered and crossed with a multiple-balancer stock; chromosomal linkage was determined, and stocks were established. Both of the transformed lines are homozygous viable and carry the P{11.8A} version of the construct. After rescue was demonstrated, theP{11.8A} transposon was renamedP{mor+11.8}. The full genotype of this construct is P{w +mC Hel89B 5′ mor +t11.8 =mor+11.8}.
Antibody preparation, affinity purification, and Western blot analyses.Antibodies were generated against MOR by inserting a 0.9-kb BamHI-EcoRI fragment into the pGEX1 expression vector (Pharmacia LKB Biotechnology Inc.), harvesting the immunogen as described previously (22), and inoculating rabbits with the immunogen in combination with Freund's adjuvant (Sigma). The antibody was affinity purified after being allowed to bind to immunoblots containing the MOR fusion protein as described in reference 25.
For Western analysis, tissues were homogenized in a solution containing 50 mM Tris-Cl (pH 7.4), 25% glycerol, 6% β-mercaptoethanol, 4% SDS, 1 mM EDTA, protease inhibitors (1 μg of leupeptin/ml, 3 μg of aprotinin/ml, 0.06 μg of antipain/ml, 1 mM phenylmethylsulfonyl fluoride, and a 1:100 dilution of protease inhibitor cocktail P2714 [all from Sigma]). The homogenates were boiled for 5 min, and the proteins in the supernatant were separated by SDS-polyacrylamide gel electrophoresis (PAGE) on 7.5% gels. The gels were blotted onto a Hybond-C membrane (Amersham) as described previously (22). The blot was incubated for 3 h with 5.0% nonfat dry milk in 10 mM Tris-Cl (pH 7.4)–150 mM NaCl (TBS). The preimmune or anti-MOR antiserum was applied at a 1:300 dilution in TBS overnight at 4°C. Following washes in TBS and TBS–0.05% Triton X-100 and a 1-h incubation in TBS with 5% normal goat serum (Biological Industries), horseradish peroxidase-conjugated goat anti-rabbit antibodies (Jackson) were applied for 2 h at room temperature at a 1:1,500 dilution. After washes were performed as described above, the immunoreactive bands were detected by enhanced chemiluminescence (ECL; Amersham).
Immunohistochemistry and in situ hybridization.Immunohistochemistry was carried out as described previously (22). In situ hybridizations were carried out by the method of Tautz and Pfeifle (46) with digoxigenin-labeled probes (Boehringer). An approximately 900-bp BamHI-EcoRI fragment extending from nucleotides 319 to 1234 of Swi3D-1was inserted into pBluescript II KS to allow synthesis of RNA probes by transcription from both the T7 and T3 promoters. Following color development, the embryos were mounted in JB4 medium (Polysciences), viewed under Nomarski optics with a Zeiss Axioskop microscope, and photographed with T-MAX 100 film (Kodak).
Coimmunoprecipitation experiments.For coimmunoprecipitations, preimmune serum or antibodies to either MOR or BRM were coupled to protein A-Sepharose beads (Pharmacia) with dimethylpimelimidate (25). The anti-BRM antibodies were obtained by immunizing rabbits with a peptide corresponding to amino acids 1617 to 1632 of BRM, coupled to keyhole limpet hemocyanin, by the use of standard procedures (25). The antibodies generated in this way recognize only one band of about 190 kDa when used in Western analysis of a crude embryo nuclear extract. Next, 15 μl of a BRM-containing embryonic nuclear fraction (see below) was added to 10 μl of beads and incubated for 2 h at 4°C in a total volume of 90 μl of HEMG (25 mM HEPES-KOH [pH 7.6], 0.1 mM EDTA, 12.5 mM MgCl2, 10% glycerol, 1.5 mM dithiothreitol, 1 mM sodium metabisulfide, 0.2 mM AEBSF, 2 mg of leupeptin/ml, and 0.7 mg of pepstatin/ml) containing 0.4 M KCl and 0.1% Triton X-100. After the incubation, the beads were washed once with a 100-fold excess volume of HEMG–0.4 M KCl–0.1% Nonidet P-40 (NP-40) and five times in HEMG–0.5 M KCl–0.01% NP-40, resuspended in SDS sample buffer, and analyzed by immunoblotting with antibodies directed against either BRM or MOR.
The BRM-containing fraction was obtained by preparing nuclear extracts from 0- to 12-h-old Drosophila embryos as previously described (52). About 400 mg of protein was further purified by heparin-agarose chromatography by standard procedures (4). The eluate of the heparin-agarose column was fractionated on an 800-ml Sephacryl S-300 gel filtration column equilibrated with HEMG–0.1 M KCl–0.01% NP-40 and developed with the same buffer (4). After a 250-ml elution volume, 10-ml fractions were collected. By Western blotting, the bulk of BRM was detected in fractions 2 to 9. Fraction 3 was used for coimmunoprecipitations.
In vitro interactions of GST fusion proteins and yeast two-hybrid analysis. 35S-labeled MOR or MOR derivatives were synthesized by using a TNT coupled rabbit reticulocyte system (Promega). To obtain MORΔNcoI and MORΔSANT, respectively, a 1,251-nucleotide NcoI fragment or a 270-nucleotideXbaI fragment was excised from the full-lengthmor pBluescript II SK cDNA while maintaining the same reading frame. To generate MORΔLEU, a 750-bp NcoI fragment was reinserted into MORΔNcoI. The large glutathioneS-transferase (GST)–MOR fusion protein employed is encoded by a 2.1-kb EcoRI cDNA fragment placed into the pGEX1 vector. GST-MOR-NH2 is the fusion protein that was used for rabbit inoculation. To generate the GST-BRM fusion proteins, genomic fragments of brm containing no introns were PCR amplified with PWO (Boehringer) from wild-type genomic DNA, using oligonucleotide primers in which we incorporated synthetic BamHI sites. The sequences of these oligonucleotides are as follows: for the large BRM fusion protein, beginning at nucleotide 742 of the brm cDNA (44), 5′ ACCAGGGATCCAGCATGCAGGACAAC 3′; for the small BRM fusion protein, beginning at nucleotide 1625 ofbrm, 5′GCAAGGATCCAAGGGAGTTCCACAGAAAT 3′; and for both BRM fusion proteins, beginning at nucleotide 2263 of the noncoding strand of brm, 5′ GGCCTTGGGAATTCGATCCTTGGCATCCTCAT 3′. The large amplified fragment was cloned into pGEX3 by partial digestion with BamHI, and the small fragment was cloned into pGEX1. Both were sequenced before use. GST fusion proteins were harvested as described previously (22).
For pulldown experiments, 50 μl of a 1:1 slurry of beads with bound protein (blank beads were used to equalize all bead volumes so that the amount of bound protein was the same in each sample by Coomassie blue staining) were resuspended in 1 ml of phosphate-buffered saline–200 mM NaCl–10% bovine serum albumin (Biological Industries) for a 1-h preincubation at 4°C with gentle rocking. Labeled proteins were added, and the incubations were continued an additional 3 h. The beads were washed four times in phosphate-buffered saline with 200 or 300 mM NaCl by centrifugation at 1,000 × g for 30 s and removal of the supernatant with a 27-gauge syringe. Sample buffer was added, and labeled proteins were visualized by methyl salicylate enhancement and analyzed with a PhosphorImager (Molecular Dynamics) to determine loading variations and the amount of retention.
For yeast two-hybrid analysis, a 2.1-kb EcoRI fragment ofmor cDNA was inserted into the LEXA202 (40) and pJG4-5 (23) plasmids and analysis was carried out by standard procedures (18, 19).
Nucleotide sequence accession number.The GenBank accession number for the sequences of the mor cDNA clones isolated in this study is AF033823 .
Isolation of new alleles of mor. An insertion of the P transposon P{lacW} into the mor region was recovered during a screen for insertion-induced mutations resulting in abdominal phenotypes, such as the loss of male-specific pigmentation or changes in the patterns of sternite bristles. This insertion produces bristles on the sixth sternite of homozygous males; the phenotype is weak and variable. It was subsequently observed that the insertion, when homozygous, enhances the homozygous phenotype ofUbx bxd-1, a mutation of the bithoraxcomplex. Since by recombination mapping it was determined to be immediately to the left of Sb, it appeared likely to be an insertion at or near the mor locus. However, test crosses with known mor alleles (mor 5 and mor 6) exhibited complementation.
The P{lacW}89B element was mobilized, and the recovered excision chromosomes were tested for viability overDf(3R)sbd 105, which uncovers the 89B region. Two chromosomes that are lethal when homozygous and when placed intrans to Df(3R)sbd 105 were recovered; both failed to complement the lethality of all mor alleles tested and of each other but were wild type over a serpentallele, srp 3. Based on these results, the new excision alleles were designated mor 9 andmor 10. However, they differ from the othermor alleles in that they fail to complement the phenotype of the P{lacW}89B insertion; males of the genotypeP{lacW}89B/mor 9 orP{lacW}89B/mor 10 display one to four small bristles on the sixth abdominal sternite.
Cloning of mor and identification of candidate transcripts.Genomic sequences immediately adjacent to theP{lacW}89B insertion were recovered by plasmid rescue and used to screen a Canton-S genomic library. Three overlapping λ clones, spanning approximately 30 kb, were recovered (Fig. 1A). A restriction-level map was derived, and P{lacW}89B,mor 9, and mor 10 were placed on the map based on genomic Southern analyses (Fig. 1B). Bothmor 9 and mor 10 are small deletions that remove the entire P{lacW} element and an additional 2 to 3 kb of genomic sequence.
Genomic sequences around the P element insertion inP{lacW}89B were employed as probes in Northern blot analyses to identify transcripts encoded by the sequence in this vicinity. Using a large probe spanning the insertion site (labeled A in Fig. 1C), three major transcripts were visualized (Fig.2A). One was an approximately 3-kb transcript (Fig. 2A) that is unlikely to encode mor since it exhibits limited expression in 0- to 8-h embryos. The second, an approximately 7.5-kb transcript (Fig. 2A and B) encoded by sequences to the right of the P{lacW}89B insertion, corresponds to the 89B-Helicase (Hel89B) gene that we have already described elsewhere (22). Hel89B is theDrosophila homolog of mot1 in S. cerevisiae (3, 13, 39) and of the human geneTAFII170/172 (11, 51). Because of the probable role of the Hel89B protein in anscriptional regulation of a large number of target genes (22), it was a good candidate for being encoded by the mor gene.
The third transcript, about 4.5 kb in size, is recognized by sequences to the left of the P{lacW}89B insertion (Fig. 2A and C). It, like Hel89B, is present at 0 to 4 h of embryonic development, reaches maximal levels at 4 to 8 h, and is found in decreasing amounts from 8 h until the second larval instar. Also like Hel89B, it is expressed in third-instar larvae, pupae, and adult male and female flies (data not shown). Upon longer exposure of the blot probed with the 2.3-kb genomic EcoRI fragment to the left of the P{lacW}89B insertion (labeled C in Fig.1C), an approximately 7-kb transcript was observed in RNA derived from 0- to 8-h embryos (Fig. 2D). Because both transcripts recognized by the 2.3-kb probe are present in unfertilized eggs (data not shown), they appear to be of maternal origin. Since the gene encoding the approximately 4.5-kb transcript was unknown to us, we set out to characterize it further.
The 4.5-kb transcript encodes a DrosophilaBAF170/BAF155/SWI3 homolog.A cDNA clone corresponding to the approximately 4.5-kb transcript was obtained from an early embryonic cDNA library, using the 2.3-kb genomic EcoRI fragment (labeled C in Fig. 1C) as a probe. This cDNA, 3.8 kb in length, was sequenced, revealing a single long open reading frame followed by 210 bp of untranslated sequence. The cDNA appears to be incomplete since it lacks a poly(A) sequence at its 3′ end and there is no stop codon upstream of the first ATG at the 5′ end.
The 3.8-kb cDNA encodes a protein of 1,189 amino acids whose sequence is shown in Fig. 3A. Comparison of the predicted protein with the data bank sequences revealed that it exhibits a high degree of homology to yeast SWI3 and even greater similarity to human and mouse SWI3-related proteins. Thus, we refer to it as SWI3D. The degree of overall identity between SWI3D and the human protein BAF170 (54) is marginally higher than that between SWI3D and human BAF155 (54) (48% identity and 58% similarity versus 47% identity and 56% similarity). The SWI3D protein is as identical to the mouse SWI3 homolog, SRG3 (28), as it is to BAF155. A lower level of overall identity is exhibited to SWI3 itself (37% identity and 47% similarity).
Alignment of the SWI3D sequence with those of BAF170, BAF155, SRG3, and SWI3 revealed that SWI3D contains all three of the conserved regions previously described in these proteins and that the level of sequence identity between SWI3D and the others is highest in these regions (Fig. 3B). SWI3D is most similar to the other proteins (especially BAF170) in region I, which is rich in prolines as well as hydrophobic and aromatic amino acids, leading to the suggestion that it may be hidden within the SWI-SNF complex (54). Region II is a tryptophan-rich domain which has been termed the SANT domain because of its presence in four proteins (SWI3, ADA2, N-CoR, and TFIIB) that participate in basal or activated transcription (1). Region III contains a leucine zipper oligomerization motif, suggesting that SWI3D may be able to bind to itself or another leucine zipper-containing protein. In addition to these conserved domains, the carboxy terminus of SWI3D is like those of BAF170, BAF155, and SRG3, very proline rich, but it is more glutamine rich than the corresponding termini of these proteins.
To isolate further-5′ regions of the Swi3D transcript, a random-primed embryonic cDNA library was probed. In this screening, an alternatively spliced cDNA, Swi3D-2(Fig. 3C), was isolated. Swi3D-2 has a candidate initiator methionine that is preceded by a stop codon. Because of the presence of a 91-bp intron in the Swi3D-2 cDNA, it is missing the sequences which encode the first 19 amino acids of SWI3D-1 and it has a different amino-terminal sequence. Unlike that of SWI3D-1, the amino terminus of SWI3D-2 is very similar to those of BAF155 and BAF170 and a little less similar to that of SRG3 (Fig. 3C).
Analysis of DNA derived from mor mutants withSwi3D probes.To ascribe mor function to either 89B-Helicase or Swi3D, we carried out Southern analyses of DNA extracted from heterozygous flies carryingmor alleles 1 through 6 over the same balancer chromosome. While no differences in the pattern of restriction fragments recognized by the 89B-Helicase probe were observed (data not shown), the Swi3D probe recognized one additional band in DNA extracted from flies heterozygous for the mor 6allele and digested with either EcoRI (Fig.4A, left panel), EcoRI plusHincII (Fig. 4A, right panel), or HincII plusXbaI (data not shown). In comparison with those of the othermor alleles, the intensity of the 2.3-kbEcoRI-generated band in mor 6 was reduced to the level of the same band in DNA derived from flies heterozygous for Df(3R)mor 7, in which themor locus is completely removed (7). In addition, a new, smaller, cross-hybridizing band appeared. This indicated that the 2.3-kb genomic EcoRI fragment that contains sequences of the Swi3D gene was disrupted in themor 6 allele.
We reasoned that the apparent reduction in size of the 2.3-kb genomic EcoRI fragment in DNA derived from heterozygousmor 6 flies could be due to an internal deletion. To localize this deletion, DNA from homozygousmor 6 embryos was subjected to PCR amplification with nested oligonucleotides that bracket this region. A 1.7-kb amplicon that was about 300 bp smaller than the equivalent genomic fragment amplified from wild-type DNA was obtained (Fig. 4B). Sequencing of the amplicon derived frommor 6 embryos revealed that it was missing 541 nucleotides of the Swi3D-1 cDNA sequence (nucleotides 2431 to 2970) and contained an insertion of 233 bp of irrelevant DNA that did not match any known sequence. Since there are no introns in this area, these alterations caused the mor 6 genomicEcoRI fragment to be 308 bp smaller than that of wild-type DNA. Thus, the mutation in mor 6 constitutes a deletion of the leucine zipper motif of Swi3D (which begins at nucleotide 2790) and of the carboxyl proline- and glutamine-rich sequences that are lost due to premature closure of the translational open reading frame. This strongly suggests that Swi3D is encoded by mor and indicates that the leucine zipper domain and the carboxy-terminal sequences of Swi3D are functionally significant in vivo.
Rescue of the mor lethal phenotype with aSwi3D transgene.A large SalI fragment, approximately 11.8 kb, that encompasses all of the genomic region known to correspond to the Swi3D transcript (Fig. 1E) was cloned into the P transformation vector pP{CaSpeR-4}. This fragment extends into the 5′ end of the transcribed region of89B-Helicase but does not include most of the coding sequence of that gene; specifically, the essential ATPase domain (3) is not included. The resulting transformation construct, pP{mor+11.8}, was injected into y1 w1118 embryos. Two transformed lines that carry the P{mor+11.8}transposon on the second chromosome and are homozygous viable were established.
One copy of the P{mor+11.8} transgene was able to rescue all heteroallelic mor 1,mor 2, mor 5, andmor 6 combinations tested (Table1). While in the absence ofP{mor+11.8} these combinations behaved as complete lethals, in the presence of the transgene they all yielded fertile progeny of the rescued class with a frequency that did not deviate significantly from that expected assuming complete rescue (χ2 test, P > 0.05). Identical results were obtained when the rescue of mor 6 was tested over the deletion Df(3R)mor 7. Given the complete rescue observed for the heteroallelic combinations, the observations that homozygous combinations of these alleles were not rescued (in the cases of mor 1 and mor 2) or showed reduced viability and/or fertility (mor 5 and mor 6) is almost certainly due to the accretion of other deleterious mutations on these chromosomes. Despite this problem, a homozygous stock ofP{mor+11.8}; mor 6flies has been established.
The lethal P excision alleles mor 9 andmor 10 exhibited complete rescue in combination with mor 6 but only partial rescue in combination with each other (Table 1). P{mor+11.8};mor 9/mor 10 adults did survive, but at a statistically significantly lower frequency (by χ2, P < 0.005 and P < 0.025 in two different crosses). Survivors exhibited reduced fertility and had a smaller wing size. Males exhibited a weak A6 sternite bristle phenotype, similar to that of the originalP{lacW}89B insertion. Rescue ofmor 9 over Df(3R)mor 7 gave very similar results (Table 1), whileP{mor+11.8};mor 9/mor 9 andP{mor+11.8};mor 10/mor 10 flies displayed more severe reductions in viability and fertility. Nonetheless, a homozygous P{mor+11.8};mor 9 fly stock has been established. Partial rescue of the mor 9 andmor 10 phenotypes by theP{mor+11.8} transgene constituted a second case in which the genetic behavior of these alleles differed from that of other mor alleles and suggested that their effects are not restricted to the mor gene (see Discussion).
We conclude that the trxG gene mor encodes SWI3D, aDrosophila homolog of proteins known to function as part of the SWI-SNF complex in yeast and mammals. In the descriptions given below, the SWI3D protein is referred to as the MOR protein. To compare the distribution of MOR with that of BRM, which is also a trxG gene product, and SNR1, which is present in a complex with BRM, and to investigate the association of MOR with BRM, we generated antibodies against MOR.
Antibodies against MOR recognize a 170-kDa nuclear protein in embryos.Polyclonal antibodies against a bacterial fusion protein containing amino acids 105 to 411 from the relatively nonconserved amino-terminal domain of MOR upstream of region I were raised in two rabbits. Both of the anti-MOR antisera, but neither of their preimmune controls, recognized a 170-kDa polypeptide when used in Western analysis of proteins extracted from embryos and adult ovaries (data not shown). Because both antisera also recognized additional polypeptides, one of the two anti-MOR antisera was affinity purified against the original immunogen as described in Materials and Methods. When used in Western analysis as described above, the affinity-purified immune serum specifically recognized a prevalent 170-kDa band (Fig. 5, right panel) that was not seen in blots probed with the preimmune serum (Fig. 5, left panel). The endogenous protein is of the same molecular weight as the MOR protein whose synthesis is directed in vitro by Swi3D-1, suggesting that the embryonic mor cDNA clone that we have isolated is complete or nearly so. The molecular mass of 170 kDa was larger than anticipated for MOR, but its human homologs, BAF155 and BAF170, also exhibited slower electrophoretic mobilities than predicted (54). An additional, faint band of about 100-kDa seen in the embryonic and ovarian extracts likely represented a degradation product of the 170-kDa polypeptide because it, as well as the 170-kDa band, was absent from extracts of late-stage homozygousmor 6 embryos selected from among their blue (i.e., heterozygous) siblings (data not shown). In these embryos, the predicted truncated protein was also not seen, probably because of its instability when it cannot be incorporated into a multiprotein complex (see below).
When the affinity-purified anti-MOR antibodies were used to examine the localization of MOR during embryogenesis by immunohistochemistry of whole-mount embryos, a tissue distribution exactly identical to that ofmor transcripts was observed. Both the transcripts seen by in situ hybridization (Fig. 6G to K) and the protein visualized with the affinity-purified antibody (Fig. 6A to E) are ubiquitous early in development and highly expressed in the central nervous system and gut of older embryos. Using these antibodies, the subcellular localization of MOR was determined. MOR was present in all of the somatic nuclei of syncytial and cellularizing blastoderm-stage embryos (compare Fig. 6B with Fig. 6A, which was stained with affinity-purified preimmune serum) but not in the posterior pole cells (data not shown). At germ band lengthening, higher levels of MOR were seen in the developing gut (the invaginating endoderm of the posterior midgut and the stomodeum) and the ventral nerve cord (Fig. 6C). In the latter tissue, the nuclear localization of MOR was still apparent. Upon germ band retraction, MOR was preferentially enriched in the mid- and hindguts and in the ventral nerve cord (Fig. 6E; compare with control in Fig. 6D) and the brain (data not shown). At this stage, no subcellular compartmentalization could be discerned.
We examined the level of MOR immunoreactivity in embryos homozygous for Df(3R)mor 7. Homozygous embryos were identified at the germ-band-extended stage, when theftz-lacZ gene of the nonhomozygous siblings is maximally expressed and when, presumably, the effects of a possible maternal contribution are beginning to wane. In homozygousDf(3R)mor 7 embryos, expression of MOR in the ventral nerve cord and brain was completely abrogated (compare Fig. 6F to the embryo of similar stage shown in Fig. 6C), further demonstrating that the anti-MOR antibodies specifically recognize the morgene product. A small amount of residual MOR immunoreactivity may be associated with the posterior midgut and the stomodeum and may represent a persisting maternal contribution of mor.
MOR is associated with BRM in embryonic nuclear extracts.The sequence similarity of MOR to SWI3 and BAF170/155 suggests that it may function as part of an SWI-SNF complex that includes an SWI2-SNF2 homolog. To test for the association of MOR with BRM, a coimmunoprecipitation assay was used on a BRM-containing nuclear fraction obtained as described in Materials and Methods. Aliquots of this fraction were immunoprecipitated with preimmune serum or with antiserum directed against MOR or BRM. The immunoprecipitates were washed in the presence of 0.5 M KCl and detergent, resolved by SDS-PAGE, and analyzed by Western blotting with either anti-MOR or anti-BRM antiserum (Fig. 7). BRM was present in the immunoprecipitate formed with the anti-MOR antibodies, but it was not present in the preimmune-serum immunoprecipitate. Conversely, MOR was present in the immunoprecipitate formed with the anti-BRM antibodies, but it was not present in the preimmune-serum immunoprecipitate. These results indicate that the two proteins are physically complexed in embryonic nuclear extracts.
MOR is able to oligomerize.To begin to examine the protein-protein interaction capabilities of MOR, we determined whether it is able to self-associate, as might be anticipated from the presence of a candidate leucine zipper domain near its carboxyl terminus. MOR was tested by the GST fusion protein interaction assay for its ability to bind to itself. In the presence of 200 mM (data not shown) or 300 mM (Fig. 8B) NaCl, full-length labeled MOR was efficiently retained on a GST-MOR fusion protein that contained most of region I and all of regions II (the SANT domain) and III (the leucine zipper domain) (16-fold greater retention than to GST alone, as judged by PhosphorImager analysis (Fig. 8A; Fig. 8B, lane 3). In contrast, MOR bound very poorly to GST alone (Fig. 8B, lane 1) or to the GST-MOR-NH2 fusion protein that included 306 amino acids from the amino terminus of MOR (less than twofold greater retention than to GST alone) (Fig. 8A; Fig. B, lane 2). Self-association of MOR was not affected by the presence of ethidium bromide at 200 μg/ml (17-fold greater retention than in lane 1) (Fig.8B, lane 7), indicating that DNA is not necessary for this interaction.
The interaction between molecules of MOR was verified independently in the yeast two-hybrid assay. A fragment of MOR identical to that present in GST-MOR was fused both to the LexA DNA-binding domain of the LEXA202 vector (40) and to the activation domain of the pJG4-5 plasmid (23). Simultaneous transformation of S. cerevisiae by both plasmids activated the lacZ of a reporter gene containing LexA binding sites, as evidenced by the blue color of colonies on X-Gal plates, while expression of either fusion protein alone did not induce blue-colony formation (not shown).
The above observations indicate that MOR is able to self-associate and that the amino-terminal sequences of MOR cannot mediate binding between two molecules of MOR. To further localize the sequences in MOR that are required for this interaction, we labeled deleted forms of the MOR protein (Fig. 3A and 8A): MORΔNcoI, which lacks the entire highly conserved leucine zipper domain and extensive flanking sequence; MORΔLEU, which is missing 38 of 90 amino acids in the leucine zipper motif and the proline- and glutamine-rich carboxy-terminal region; and MORΔSANT, from which half (56 amino acids) of the SANT domain, as well as 37 somewhat-less-conserved residues N terminal to it, has been deleted. The MOR derivatives were tested for retention by the immobilized GST-MOR fusion protein. Removal of half of the SANT domain in MORΔSANT did not appear to affect the ability of MOR to homooligomerize (14-fold greater retention than binding of full-length MOR to GST alone) (Fig. 8B, lane 6). (Note that slightly less radiolabeled MORΔSANT was loaded onto the GST-MOR fusion protein [Fig. 8B, lane 11], thus largely accounting for the smaller amount retained.) However, self-association was severely compromised by removal of the extended leucine zipper domain in MORΔNcoI (only fourfold greater retention in lane 4 than in lane 1). Reintroduction of part of the leucine zipper motif in the MORΔLEU construct appeared to restore some of the binding activity (sevenfold greater retention) (lane 5). These data indicate that MOR is capable of forming homooligomers and that region III, the leucine zipper motif, is likely to contribute to this interaction.
MOR binds to BRM.MOR was tested for its ability to interact with BRM, with which it is associated in embryonic nuclear extracts. In these experiments, we focused on domain II of BRM because deletion of this region causes a decrease in the size of the BRM complex, presumably due to the loss of one or several subunits (16), and because yeast two-hybrid analysis revealed an interaction between this domain of SWI2-SNF2 and the SWI3 subunit of the yeast complex (47, 48). While domain II of BRM consists of residues 549 to 610, the two GST fusion proteins that were generated for the interaction assay were more extensive and included amino acids 230 to 736 or 524 to 736. At 200 mM NaCl, labeled full-length MOR was retained on the immobilized GST-BRM fusion protein containing residues 230 to 736 approximately as well as it was retained by immobilized GST-MOR (12- and 13-fold-greater retention than of full-length MOR to GST alone) (Fig. 8C, lanes 1 and 3). In contrast, binding of MOR to beads bearing the same amount of a GST-BRM fragment containing only amino acids 524 to 736 was much less efficient (sevenfold greater retention) (lane 6). Thus, MOR is able to interact with BRM and this association can be mediated by 507 amino acids in BRM that include domain II.
To examine the role of different protein domains of MOR in binding to BRM, we tested the ability of the MOR deletion constructs to be retained by the BRM fusion proteins. MORΔNcoI was efficiently retained by GST-BRM 230–736 (15-fold greater retention in lane 4 than in lane 2), implying that the association of MOR with itself or another leucine zipper-containing protein is not necessary for its binding to BRM. This is not the case for MORΔSANT, whose binding to an equivalent amount of the large BRM fusion protein was significantly reduced relative to the binding of full-length MOR (only fourfold-greater retention in lane 5 than in the control). While MORΔNcoI was retained, albeit poorly, by the smaller BRM fusion protein (sixfold-greater retention in lane 7 than in lane 2), the level of retention of MORΔSANT fell almost to that of the negative control (twofold-greater retention than binding of the full-length protein to beads carrying GST alone) (compare lanes 8 and 2). These results suggest that the SANT domain of MOR may play a role in the association of MOR with domain II and adjacent residues of BRM.
The fruit fly BAF170/BAF155/SWI3 homolog is a member of the trxG proteins.We have isolated a Drosophila homolog of the yeast SWI3 gene and the human BAF170 andBAF155 genes and demonstrated that it is encoded by the previously known trxG locus mor. Its identification asmor was achieved by Southern analyses of DNA derived from flies with mutations in mor, by characterization of a genomic Swi3D fragment derived from homozygousmor 6 embryos, and by rescue of themor phenotype. The origin of mor 6 by γ-ray mutagenesis (29) is consistent with our observation that it encodes an altered form of mor in which 541 bp have been deleted and a 233-bp insertion of foreign DNA has occurred, leading to loss of the leucine zipper motif as well as the carboxy-terminal proline- and glutamine-rich sequences.
Genomic DNA encoding the Swi3D gene is able to completely rescue the lethal phenotype of mor alleles 1, 2, 5, and 6. In contrast, introduction of the entire neighboring89B-Helicase cDNA into the background of themor 6 allele in combination withmor 9 is completely inefficacious in rescuing the heteroallelic lethal phenotype (55). Thus, the 5′ portion of the 89B-Helicase gene included within theP{mor+11.8} construct is not mediating the observed rescue. On the other hand, the residual phenotype observed in flies carrying the mor 9 andmor 10 alleles after introduction ofP{mor+11.8} is likely to be due to the effect of these mutations on the 89B-Helicase gene rather than failure of the transgene to completely compensate for themor deficiency. We interpret this to be the case because the89B-Helicase transgene appears to be able to rescue the bristle phenotype that is associated with P{lacW}89B(55). It will be interesting to test whether the introduction of both transgenes results in the complete rescue of themor 9 mor 10 phenotypes.
MOR is a component of the Drosophila counterpart of the yeast SWI-SNF complex.We have observed that theDrosophila BAF170/BAF155/SWI3 homolog, encoded bymor, is physically associated with BRM in embryonic nuclear extracts. The presence of an identical protein, named BAP155, in a highly purified 2-MDa BRM complex isolated from Drosophilaembryos was very recently reported by Papoulas et al. (36). Significantly, analysis of the eight subunits of the BRM complex revealed that MOR/BAP155 is the only component, other than BRM and the previously identified SNR1 (14), that is encoded by a trxG gene. Two additional trxG proteins, ASH1 and ASH2, were found to be present in distinct high-molecular-mass complexes.
The identification of mor as a gene encoding a component of the Drosophila BRM-containing complex provides a biochemical basis for the close functional relationship between mor andbrm, both strong and well-characterized trxG member. Indeed, the phenotypes resulting from mutations in each of these two genes exhibit many similarities. For example, alleles of both morand brm were repeatedly isolated in genetic screens for dosage-dependent modifiers of dominant mutations in Pc(29); mutations in either gene affect transcription of multiple homeotic genes and the segmentation gene,engrailed; both of them are required for oogenesis; and both may be involved in cell viability of imaginal disc cells but not abdominal histoblasts (6, 7, 16, 44). More recently,mor was the only trxG gene found to interact with a dominant-negative brm transgene (36). Further analysis will be required to clarify the relationships of the trxG genes that are not components of the BRM complex to MOR and BRM.
Comparison of mor gene product distribution with those of brm and Snr1. The expression pattern ofmor overlaps with those of brm andSnr1 but is more widespread. The presence of mortranscripts in unfertilized eggs, as determined by Northern analysis, suggests that there is a maternal contribution of the morgene product, as has been reported for brm andSnr1 (6, 14). In addition, mor, likebrm and Snr1, is most highly expressed in young embryos up to 8 h of age (14, 15). Unlike those ofbrm and Snr1, mor transcripts are also present in adult males. Although brm transcripts were not detected in adult males, low levels of BRM protein have been reported (16).
During early embryonic development, mor transcripts and the nucleus-localized MOR protein exhibit a ubiquitous distribution like that of BRM and SNR1 (14, 16). After germ band retraction, they are observed at high levels in the central nervous system (CNS) and in the mid- and hindgut. This contrasts with SNR1, which is almost exclusively found in the ventral nerve cord and brain at the end of embryogenesis (14), and BRM, which is ubiquitously expressed and only somewhat localized to the CNS (16). Expression ofmor transcripts and protein in the endodermal primordium is consistent with the midgut abnormalities described in mormutant embryos (7) and suggests that MOR is functional in this tissue. It will be interesting to determine whether MOR is more stable in the cytoplasm, since in Df(3R)mor 7embryos what we assume to be a small amount of residual maternally derived MOR protein appears to be preferentially retained in the midgut and stomodeum, tissues in which nuclear enrichment of MOR has not been observed.
Protein interactions of MOR.The protein-protein interactions that stabilize the SWI-SNF complex are not well understood. Some of the direct associations that have been reported are binding of TFG3/TAF30 and SNF5 (10) and an interaction between SNF11 and SNF2 (47). Because of the presence of a putative leucine zipper motif in MOR, we examined the ability of MOR to self-associate, using a GST fusion protein interaction assay and the yeast two-hybrid system. Our data indicate that MOR is able to oligomerize in vitro and in vivo in the yeast two-hybrid system. In addition to demonstrating an in vivo interaction between two molecules of MOR, our results indicate that a MOR derivative containing most of domain I, all of domains II and III, and most of the proline- and glutamine-rich tail cannot activate transcription when artificially tethered to DNA. This could be due to the absence of some essential MOR sequences or to the inability of the fruit fly protein to nucleate assembly of the entire yeast complex. By comparison, SWI2-SNF2, SNF5, and SNF6 all function as activators when targeted to a promoter as LexA fusions (32, 33).
Neither region I nor the SANT domain has to be complete for self-binding of MOR to occur. Since oligomerization is sensitive to the extent of removal of the leucine zipper domain, our results strongly suggest that the leucine zipper motif of MOR contributes to the ability of this protein to self-associate in vitro, but they do not eliminate the possibility that the carboxy-terminal region is involved.
The demonstration that MOR is able to self-associate raises the possibility that it is present in two copies in each complex, similar to BAF170 and BAF155, which are both present in each human complex (53, 54). These results support a dimer-like model for the structure of the SWI-SNF complex, with duplication of some or all subunits. Such a model has been proposed previously because the overall molecular mass of the complex is much greater than the sum of its individual components (54). It is also possible, however, that in vivo, the leucine zipper motif of MOR is involved in an association between MOR and a different leucine zipper-containing protein. In either case, the embryonic-lethal phenotype resulting from deletion of the leucine zipper and carboxy terminus in the polypeptide encoded by the mor 6 allele suggests that these domains are functionally required in vivo. Robust assembly into the BRM-containing complex via interactions involving the leucine zipper motif may affect the stability of the MOR protein, since we are unable to visualize the truncated protein whose synthesis is directed by themor 6 allele. This is similar to the reduced stability of SWI3 that was noted in the absence of SWI1 and SWI2 (38) and may also derive from the failure of SWI3 to be incorporated into a complex.
We tested whether MOR, which is complexed with BRM in embryonic nuclear extracts, is able to interact with this protein in vitro. The data indicate that such an interaction can occur and that it involves domain II of BRM, as well as adjacent residues on the amino-terminal side of this domain. The motif in MOR which may mediate the interaction with BRM is the SANT domain. The possible role of the SANT domain is suggested by the significant reduction in the binding of MOR to BRM when 56 amino acids of this domain are deleted, but a role for an additional 37 non-SANT domain residues that were also removed cannot be excluded. Unlike the related DNA-binding domain in the MYB family of proteins, the SANT domain in BAF170 was not shown to have detectable DNA-binding activity in gel shift assays (54), and its function is unknown. These findings, for the first time, ascribe a possible role for the SANT domain in protein-protein interactions.
We thank Daniel Jay, in whose laboratory portions of this work were carried out; James Kennison, who provided moira stocks; Yossi Markson for limitless patience in preparing the figures; Simon Greenberg, who assisted in DNA analysis; and Benny Shilo for reading the manuscript.
M.A.C. was supported by a grant from the National Science Foundation; N.B.Z. was supported by grants from the Israel Cancer Research Fund and the Israel Academy of Science.
M.A.C. and C.M. contributed equally to this work.
- Received 24 August 1998.
- Returned for modification 1 October 1998.
- Accepted 29 October 1998.
- Copyright © 1999 American Society for Microbiology


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Three or four times a week I get an email inquiry from consumers who have read my prior posts on buying REO's. (See the most popular of these posts at: http://therealestatewhisperer.blogspot.com/2008/08/reos-everything-you-need-to-know.html or http://therealestatewhisperer.blogspot.com/2008/12/types-of-sales-seen-in-todays-market.html). Specifically the email requests are for an addendum that I reference in these posts.
I am always hesitant to give it directly to the consumer. Laws vary greatly from location to location, and I don't want to be accused of giving real estate advice in areas where I am not licensed. Each bank addendum is different, and therefore the addendum should reflect the language specific to the rest of the contract. The addendum is intended to serve as an example, and not a form that should be used carte blanche in all REO transactions. Then, I feel bad when I tell a consumer to have their agent contact me directly to obtain the addendum. I feel worse when I am overwhelmed carrying for my own business and drop the ball and fail to send it for them; and I confess that I've done that a couple of times inadvertently. I fear it will be more common, as I've switched primary computers and it is a difficulty for me to go to the other computer to forward the addendum.
You see, I am using the addendum less because in our local market, traditional sales are returning (Hip, Hip, Hooray), short sales are becoming more successful and I am simply working less in REOs. I currently have one REO listing - hits the market today; but all of my deals that are under contract for the moment include contracts for purchase in traditional sales or new home sales. A new day is dawning in Northern Virginia.
In any case, I think I've come up with a solution; as I know some areas are just now being hit by floods of REOs and you really, really need this! I found a place where I can store this addendum online and make it available to you. Here it is:
http://h1.ripway.com/vchrisner/Addendum to Contract - for reos.doc
Remember, reading blogs and educating yourself is great, but does not replace competent real estate or legal representation - ever. There are just too many variables; and a blog can not possibly address them all. However, the information you get from my blogs, and others, should serve to stimulate educated discussions between you and the professionals you are choosing. You SHOULD NOT know more than your agent. If you do, you should get another one. I firmly believe the value of an agent is advice. Sure, there are service related conveniences - access to listings, lockboxes, forms, affiliated companies that may offer free or low cost services, etc. But, what a Real Estate Agent should bring to the table, above all else, is their knowledge.
Now, go forth and purchase!!! And, if you are in my area - look me up! I'd love to serve as your chosen real estate consultant. www.VickyChrisner.com 703-669-3142 email@example.com

subset: commoncrawl


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New York — During a High Level Meeting on Ebola at the United Nations on September 25, UN Secretary General Ban Ki Moon called for a heightened response from the international community. "The world can and must stop Ebola – now," he said. One of the participants was Nkosazana Dlamini-Zuma, who leads the African Union Commission, the secretariat of the 54-member organization. In a brief interview, she addressed the need for assistance and what the AU is doing to assist countries most seriously impacted by the outbreak.
What are the most important steps that need to be taken now?
I think that we should understand that the problem of Ebola is twofold. Nigeria was able to contain it because they have strong institutions and strong public health responses where possible. Countries that are doing their best to come out of conflict and are still building their institutions and strengthening their capacities, they were not able to give as full a response as Nigeria did. So it's very important to ensure that their capacity is strengthened.
That's why the African Union is mobilizing young healthcare workers – laboratory technicians, nurses, doctors – to go to these countries to strengthen their brothers and sisters who are on the ground. We've already sent a team to Liberia. We're going to send a team to Sierra Leone and to Guinea to ensure that they have adequate protective materials so that those who are attending to the ill are not also infected.
It's very important that countries show their solidarity – open their borders, resume flights, and remove travel bans because these countries need all of the support that we can give them. People panicked because it was the first time we had an epidemic of such proportions, and they took some extreme steps. I think now that that is going to be reversed. Some places have already reversed it. That is very important.
It's also important that beyond the epidemic itself, we must look at the socio-economic impact of Ebola – food security for instance. People are not planting. The movement of food is not what it used to be. I hear that prices have gone up for food. We have to see how we can support these countries beyond the epidemic itself. They will need a lot of support from all of us. Their GDP has already been revised downward, so this will have great impact beyond the epidemic.
Do you think the International response has been sufficient?
I think it is beginning to be that. It is beginning to build up, but we need more. Not just financial, but resources, logistic support, protective clothing, people who can assist professionals.
What is the level of support the AU is giving?
That's what the AU is doing. We already have some things in Liberia, we are going to be sending more to Sierra Leone and Guinea. Because we are constrained with financial resources at the moment, we are talking about sending 120 or so. About 40 to each of the three affected countries. We've already sent 28 to Liberia. These are laboratory technicians, who are going to be on the ground assisting nurses and doctors. We need more, since we we'll have to rotate them, because they can't be there forever. If we get more resources, we'll send more.
Has there been a special appeal to African Heads to state to assist the Ebola fight?
We're asking them for three things. The first one [is] to stop travel bans. Two – to assist financially or three – through concrete assistance – personnel, laboratories, protective gear. Beyond the epidemic, we'll look at how we can continue to work with these countries to strengthen their health services, but also to assist in terms of the socio-economic impacts that they are going through.
It is very important to open borders, restart flights and stop the travel bans. This is having an impact on the people, not just socio-economic, but also morale. And we're calling not only on governments, but also private citizens. Everyone can help.


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Date: October 26, 2016
Time: 1:00 EST
Price: FREE
Navigant Research estimates that utility companies will spend almost $50 billion on asset management and grid monitoring technology by 2023. Today many organizations are facing budgetary challenges in order to increase reliability, uptime and safety within their facilities.
The industry is adapting to new technologies including utilization of advanced sensors and sensor fusion, edge devices, artificial intelligence, and machine learning to create the maintenance center of the future.
Bernie Cook, former Director of Maintenance and Diagnostics at Duke Energy and now VP of Woyshner Service consulting, will join us to provide practical guidance and examples of how utilities can begin adapting these next generation technologies within their facilities to drive significant reduction in maintenance costs.
Following Bernie, Stuart Gillen, Director of Business Development at SparkCognition, will give examples of how machine learning technologies are augmenting current practices that make maintenance engineers more efficient at predicting critical asset failure.
Join this webinar to learn about:
- Real examples of ways utilities are moving to more advanced monitoring and diagnostic capabilities and the technologies involved.
- How machine learning can improve equipment reliability and performance, and reduce operational and maintenance costs.
- How machine learning can augment or even supplement human subject matter experts by providing significant advance notice of asset performance issues.
Bernie Cook
Executive consultant
Duke Fossil & Nuclear
Stuart Gillen
Director, Business Development
Jon Arnold
Residing in Austin, Zpryme is a research, media, and events agency with a focus on energy. Based on the creative direction of our associates Zpryme, produces influential research, Energy Thought Summit (ETS), and premium branding experiences that challenge the status quo.


text:
EF União
Escola de Futebol União.
Ilha do Sal, Cabo Verde
Download the vector logo of the EF União brand designed by Maurizio Vieira in Encapsulated PostScript (EPS) format. The current status of the logo is active, which means the logo is currently in use.
- Designer:
- Maurizio Vieira
- Contributor:
- Maurizio Vieira
- Vector format:
- eps
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- Active Report as obsolete
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- Updated on:
- Wed, 11/06/2013 - 16:54


text:
Endless Life
(Literary Masterpieces, Volume 14)
Download Endless Life Study Guide
More than Allen Ginsberg, Sylvia Plath, Anne Sexton, or even Rod McKuen, Lawrence Ferlinghetti was the poet of the 1960's, the poet who spoke to the youth of that generation, like them full of hope and idealism, believing in love and sexual freedom, rejecting war, mechanization, and dehumanization. His words were used on banners, in demonstrations, even in church services, and they summed up the feelings of thousands of young people.
Endless Life gives a retrospective of Ferlinghetti's work over the past twenty-seven years since his first book, Pictures of the Gone World (1955), which established once and forever his characteristic style and voice. His free verse lines floated back and forth across the page in no special form, suggesting fragmented impressions, often allowing for interesting juxtapositions of images in adjacent lines. His voice combined classical poetic diction (often incorporating quotations from William Shakespeare, the Romantic poets, and later Americans) with colloquial language that often involved clever wordplay—puns, unexpected word choices, and epithets that united two figures of speech in a single new one. His characteristic themes appeared: pleasure in the here and now (shown through vignettes that captured mundane incidents and revealed in them something special, spiritual, even mystical), joy in sexuality and appreciation of the body and all physical sensations, ridicule of American commercialism, and criticism of the injustices and abuses that prevail throughout the nation and the world.
"The World Is a Beautiful Place" is an excellent example of his method. It uses several loose stanzas which begin with a repetition of the title, followed by various explanations and qualifications which often deflate the praise by recognition of some of the less beautiful aspects of life, presented through wordplay: "its men of distinction/ and its men of extinction," "its various segregations/ and congressional investigations/ and other constipations." The poem culminates in a simple but exhuberant listing of enjoyable human activities, interrupted at last when "right in the middle of it/ comes the smiling/ mortician." The fact of death does not so much negate the value of what has been described as it adds to it a deeper awareness.
Ferlinghetti's subsequent collections continued to give evidence of his playfulness with language and his enjoyment of the world around him. In these later collections, however—beginning with his second and most popular (with almost a million copies in print the world over), A Coney Island of the Mind (1958)—he took on a stronger persona as a prophet, such as one of his favorite poets, Walt Whitman. His goal was to awaken and enlighten his fellow Americans, not only to the beauties of their world but also to their repressed selves and to the corruption and alienation rampant in their world. He often sought to do this in an incantatory style, recalling Whitman's psalm-like cadences and long catalogs.
Perhaps his greatest poem in this mode is "I Am Waiting," a poem that retains its validity and lighthearted but serious appeal a quarter of a century later. Its feeling of perpetual expectation for the miraculous to occur emerges from Ferlinghetti's use of clichés which have permeated American culture, here twisted or taken literally, with the eternal optimism of the American spirit. Like most of his longer poems, "I Am Waiting" uses short lines all starting at the left margin to give emphasis ot the repeated phrasing. This poem's expectations of an apocalyptic "rebirth of wonder" incorporate both the whimsical hopes typical of simpleminded patriotic fundamentalism ("I am waiting/ for them to prove/ that God is really American") and more serious dreams that are hardly more conceivable ("I am waiting/ for a way to be devised/ to destroy all nationalisms/ without killing anybody"). All these wondrous events epitomize the spirit of hope and dreaming...
(The entire section is 1,892 words.)


text:
Spirit and in Truth
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Perhaps you have at some time spoken out loud to yourself. No, it does not mean that you are losing your sanity. However, it may mean that you better remember something important if you hear yourself say it out loud. A similar principle is in play at the Fifth of Ephesians. At Verse Nineteen, the apostle Paul tells us that we are to be speaking to ourselves in the psalms, hymns, and spiritual songs; in other words, we are to be hearing and listening to what we sing, and it matters a great deal what is said. The Third of Colossians, Verse Sixteen, tells us that it is with those same hymns that we teach and admonish one another. It matters what the songs say because it matters what is taught by the singing of them. If the word of Christ is dwelling in us in all wisdom as Paul said it should, then we are well able to discern whether or not a song or hymn is offering the proper teaching and admonishment. The singing is really less about keys, notes, sharps, and flats. It is more about worshiping in spirit and in truth.
Written and voiced by David Hayes Prophater
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text:
Mathieu Bastareaud is excited by the prospect of Julian Savea making a high profile move to Toulon.
The prolific Hurricanes wing, fourth on the list of New Zealand's all-time leading try-scorers, is reportedly on the verge of signing a two-year deal with the Top 14 side.
Savea has not played for the All Blacks since facing the British and Irish Lions last year and does not appear to be in Steve Hansen's plans.
The 27-year-old flyer is said to have agreed to move to France at the end of the Super Rugby season, which is music to the ears of Toulon captain Bastareaud.
"This is big news for us, one of the best wingers in the world, I'm just happy for the club." said the centre, who will lead France out in the second Test against the world champions at Westpac Stadium on Saturday.
Savea has scored 46 tries in 54 Tests for the All Blacks.
KEO.co.za News wire is powered by opta


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not to burst your bubble but they said they will NOT be making any other additional sizes for it. It is stuck in that configuration forever.
I think the antique bronze finished hardware is so sexy and elegant on that black on black finish!
It's a beautiful kit and I bet the European Maple sounds amazing.
I do wish though they would have also done something one off special for it like a one off hardware or 12mm rods like the DW vs 10.5mm you know?
Something subtle but one off you can't get on anything else other than just changing the color.


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Kentucky State Apiarist
The Kentucky State Apiarist identifies and eradicates infectious disease in honeybee colonies. In addition, the State Apiarist helps to educate the non-beekeeping public about the importance of honeybees, and offers best advice practice to the beekeeping community. Hive inspections are available with an appointment, and health certificates are provided for the transport of bees out of state. The State Apiarist is also available to speak at beekeeping meetings and various other functions.
Pollinator Protection mapping program
The Pollinator Protection mapping program is now available to beekeepers and pesticide applicators. If you would like to use this program to register your apiaries and/or pesticide spraying applications, please use Chrome, not Internet Explorer. The link is provided on the sidebar. You must register as a user first, then register your apiaries or applications. A video is provided to help you with this process.
Honey Label Review
Beekeepers should send all labels for review in a pdf format for generating print-outs. JPEG files are not recommended because these files are not as printer-friendly with the office equipment for printing out labels. Contact information is listed under the Honey Label Review.
Asian Giant Hornet Information
Asian Giant Hornets are not in Kentucky, but the Kentucky State Apiarist has set traps to monitor for the appearance of the Asian Giant Hornets.
New Pest Response Guidelines
Pest Alert: Asian Giant Hornet
Standard Operating Procedures for Reporting a Pesticide Kill to Division of Environmental Services
If you suspect a hive has been impacted by pesticide applications, please adhere to the following standard operating procedures that KY Dept. of AGR maintains when collecting samples:
- First, after beginning a routine inspection, if you suspect that a hive has been impacted by chemicals, go no further and put the hive back together. Leave the area alone and immediately contact KY Dept. of AGR inspectors to arrange a meeting at the following number 1.502.573.0282.
- Do not take samples. The KY Dept. of AGR inspectors cannot accept samples of bees collected prior to their investigation.
- The investigator will take a statement from the beekeeper about the possible hive-contamination event. It would assist in the process if the beekeeper has supporting evidence such as photographs of the impacted hive before the chemical spray, video footage, pictures of actual spraying, etc. These items are not mandatory, but highly recommended.
- As part of preparing a statement, the beekeeper should have the contact information of their neighbors and suspected sources of contamination, the types of plants in bloom in neighboring fields, where potential water sources may be, date of alleged chemical sprays, as well as any information that can help the inspector assess the situation. Being able to brief the KY Dept. of AGR inspector with this information expedites the process of recording a statement.
- Since inspectors may not be beekeepers, beekeepers should have an extra jacket, a smoker, and be willing to collect samples if the inspectors are either allergic or apprehensive about working a beehive.
Hive and Apiary Inspections
The State Apiarist's Office provides free hive and apiary inspections for Kentucky beekeepers. The State Apiarist will inspect apiaries and hives for the presence of honeybee diseases and pests. Specimens for laboratory examination will be collected if needed. After the inspection the beekeeper will be provided a written report listing any diseases, pests or other problems found and a listing of remedies for correcting these problems. (See The Importance of Late Summer/Early Fall Inspections).
General Policy for Apiary and Hive Inspections:
- Beekeeper must be present during requested inspections.
- Beekeeper will open hives and remove frames for inspections.
- Attempt will be made to conduct inspections as soon as possible after request but inspection dates and times subject to State Apiarist's schedule. To schedule an apiary and hive inspection, or for more information about this service contact our office.
Grant Opportunities
Start here to find grants that will help you get your beekeeping operation off the ground or take it to the next level:
Kentucky Agricultural Development Fund investment area guidelines
Kentucky Proud Programs
Bee-friendly Wildflowers
Here's a sample plan to show you how affordable it is to raise a bee-friendly wildflower garden.
Kentucky Beekeeping Association Listing and Contacts
Kentucky's beekeeping associations are a great source of beekeeping information and assistance for Kentucky beekeepers. Beekeeping associations hold regularly scheduled meetings and other activities. Contact the individual beekeeping associations for more information (see individual listings). Please check this page often for more information as we will update regularly.
- President: Mike Mabry
- President-elect: Tom Ballinger
- Vice-President: Joel Gonia
- Secretary: Joe Chang
- Treasurer: Wes Henry
- Webmaster: Rick LeMarr
- Past President: Chris Renfrow


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Today, I have with me author Helen Stringer, author of Spellbinder and The Midnight Gate, two wonderfully spooky and supernatural middle grade novels. I just reviewed Spellbinder last night and will be reviewing The Midnight Gate later this week, both are spectacular middle grade novels, with a great combination of funny commentary and seriously spooky moments.
I asked Helen if she would talk a little about where she got the inspiration for some of the creepy places that her novel take place in.
One of the things that has always got my imagination firing on all cylinders is places. Old places, new places, buildings, countryside, towns, cities – it really doesn't matter. Most of the time, particularly with historical sites, the only view you are going to get is as part of a tour group. Often this is fine – when I was at school we were taken to an old Elizabethan house outside Liverpool so many times that the actual tour blurb is engraved on my memory. The house is called Speke Hall, which became Arkbath Hall in Spellbinder, where the tour guide that takes Belladonna and her classmates through the house spouts, verbatim, the old Speke Hall tour of my childhood!
Many real places pop up in Spellbinder and Midnight Gate, an old monastery near Barrow in Cumbria, a 1930s apartment building in Liverpool, and an electric store/vaudeville theater in Los Angeles, as well as my old school – Belvedere, which really is made up of three Victorian Houses joined together. The one place that I grew to know almost as well as my school, however, is the fabulous Croxteth Hall.
Croxteth was the home of the Earls of Sefton. It is a vast pile right on the edge of Liverpool and was such a mish-mash of styles that after the last earl died the National Trust turned down the opportunity to run the property. Enter the city council. They took on the old place and when the surviving countess gave permission for them to have the family papers, I was hired to catalog them. This was my first job. It was supposed to be for six months and I was totally unqualified, which didn't matter as I was only supposed to be making lists. Then the first of two disasters struck. First, the countess changed her mind about the papers (oh, no! I'm going to lose my job!), then the local paper ran a campaign questioning why the place wasn't open to the public (hooray! I get a new job!).
The scramble was now on to prepare the place for visitors. There was only one problem: the countess had sold off nearly all the furniture, so the first part of my new job was to research the family history and put together a tour with what we had to hand. The second was to actually do the tours at the weekends.
It was kid in a candy store time! A bunch of my friends were hired to help do the tours (what were the managers thinking?) and during the week I spent most of my time either downtown in the central library reading the personal diaries of the lady who was countess in the early 1900s or wandering around the huge house, keys in hand, seeing what they would open. The answer to that was…everything!
We meandered all over the place, found the earl and countess's ceremonial robes, complete with coronets (and tried them on, of course), found boxes and boxes of photographs taken by the same countess who wrote the diaries (she was a keen photographer and had a dark room at the top of the house), and explored the absolutely massive kitchens.
The kitchens consisted of a giant room with dozens of stoves and huge worktables, as well as smaller rooms for the preparation of vegetables, meats and confectionary. (This is the kitchen that Belladonna finds in the House of Mists in Spellbinder.) The kitchen also featured a large dumb waiter that was powered by water. The servants would simply load up the dumb waiter and pull an enormous lever. There would be the sound of rushing water and the huge box would move slowly upwards to the dining room. Needless to say, we had to experiment with this. One of my friends climbed into the box, we closed it, pulled the lever and ran up the servants' stairs to the small ante-room next to the dining room. It seemed to take forever, but eventually the box hove into view and we got our friend out. His name was Steve, which is odd, because it's exactly the kind of thing Steve Evans would do! (I should probably pause here and point out that it was really dangerous and could have gone wrong in so many ways. But it didn't. Heh.)
By the end of the summer I had worked seven days a week for almost a year. I was exhausted but had absorbed everything about the old house and grounds and it has never left me: the perfect proportions of the eighteenth century rooms that make you feel calm as soon as you step into them, the winding servants stairs and basement passages, the grand main staircase and pillared balconies, the tiny servants' bedrooms at the top of the house, and perhaps most of all the details of the life of the daunting woman whose domain it was in the days of balls and hunts and garden parties.
These days the house is run like a well-oiled machine, with a proper visitors center and glass cases of artifacts. But I prefer to remember it as it was – a chaotic playground and an inspiration that I find myself returning to again and again.
- The Monster Report - May 17, 2020
- Book Review: One Last Shot + Giveaway - May 14, 2020
- Book Review: Air Awakens - May 13, 2020


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With cbd-medicatie.nl we want to provide you with factual information on CBD and other cannabinoids that are of interest. We strive to be as transparent as possible. For this reason references to scientific articles can be found in all sections of this website regarding physical and mental ailments. We encourage you, if you are interested in a particular topic, to also read the literature on which our articles are based. Often the full text off these articles can be found online, or at least the abstract is available. You'll find a list of web links at the bottom of the page.
Apart from scientific literature, we have made good use of some (hand) books, articles and websites. In particular, the Handbook of Cannabis, edited by R. Pertwee, has been a very important source. Below is a full list of sources used by us.
Pertwee, R.G. (2014). Handbook on cannabis. Oxford: Oxford University Press
Cervantes, R.C. Clarke, C. Conrad, A. Jansen, R. Mechoulam, M. Guzman, D. Richard, Lucas, D.E. Wirtschafter (2015). Medical cannabis: From marijuana to synthetic cannabinoids. Paris: Mama Editions.
Magazine articles


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Brand New! Has original tags. This will be great for you and you horse and I hope you get good use out of it!
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Michal Golan Prismatic Collection Heart Shape Post Drop Earrings. Set in brass electroplated with 24k gold and/or sterling silver. The earrings measure approximately 1 1/4 inches long by 7/8 inches wide. Designed and made in the USA in New York City.
About Michal Golan
Michal Golan is an Israeli born, New York City based jewelry designer. In her studio Michal creates hand worked jewelry pieces combining the look of the antique with contemporary fashion sensibility. The jewelry is 24k gold or sterling silver electroplated over brass or white metal. Semi-precious stones, freshwater pearls, Swarovski crystals, and glass are the components.


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[amazon_link asins='B00XANZZTA' template='ProductCarousel' store='engineerdiari-21′ marketplace='IN' link_id='2796422f-3cc2-11e7-82d3-ab185a88311d']So, yesterday CEO of Google Sundar Pichai along with senior product managers in Google presented eleventh Google I/O – Google Keynote.It was 2 hours long event where Google disclosed all the progress made since last year, and also their vision for the coming year. So, if you haven't watched it then covering below are some highlights of the event.
1. OUTLINE:The event outlined all the major Google products- Search Engine, G-mail, Android, Youtube, Maps, Google assistant, Google drive, Google photos and much more but they major advancement in all these products is the shift from mobile first to AI first. That is Google is making its products smarter by shifting them to technologies such as Machine Learning and Artificial Intelligence.Now lets' have a look at key highlights at all the other products.
2.Google lens: Incorporating Machine Learning , Google assistant and search engine all in one to take basic search engine at another level. So, Google lens is indeed pretty cool and definitely could be the next big thing for Google.
3.Google for Medical: Don't worry this is no product, just an heading. Google is now investing a lot of money to use technology help the world in medical arena just like Bill Gates and Mark Zuckerberg and diabetes curing and detection of breast cancer being the top most priorities. In the end of the keynote Sundar Pichai highlighted achievement of 17 year teenager for breast cancer using Machine Learning.
4.Google Assistant: This year Google is investing a lot in taking Google Assistant to another level which could be understood by knowing that almost 20% of keynote talked about it. And Google assistant is taking the dream of a helper Robot who lives in our mobile alive and way- way better than Siri!
5.Google home and Google photos: Google home is a speaker with built in Google Assistant to provide the same experience but this time our Robot lives in a speaker . Google photos is taking the way we store our photos to another level too with suggested sharing being at top of it.
6.Youtube and Android: The most important update to be included in Youtube is Superchat and its no exciting or new at all. And in android lot was talked about getting android in the wearables and TV, which have started witnessing now only, but the big thing is that Android would now support another language called Kotlin. I still don't know why its so big?
7.Google play store and Android O: Google is increasing the security level in apps, but the big announcement here was of launching cheaper phones having Android O.This step is worth the biggest applaud as world indeed needs smarter phones at cheaper rates and Google explained all regarding the low processor and development of Android O.
8.Virtual Reality/Augmented Reality: For all the game lovers and education department this is the big highlight. Samsung Galaxy S8 would support virtual reality experience. And, also google showed us some pretty cool advancement in Visual Positioning System to find anything in our home via phone!
9. Google for jobs: The biggest announcement here, of Google helping employers find employees and working with Linkedin and monster.com too.
Summary: It was amazing to see so many women in the software world and doing at par with men.And also being Indian seeing half of the software leaders as Indian in indeed pretty special. For all the engineers and developers out there, one thing we must take from here is that Machine Learning And Artificial Intelligence is being used everywhere and we must include them in our skill set for better future!


text: Would you sign Vinicius if it means losing Jimenez? Yes Vote No Vote Related Polls Are Wolves a selling club? Is Leander Dendoncker part of Nuno's first XI? Is Raul Jimenez as good as Harry Kane? Can Wolves break into the top four in the next two seasons? Related Articles Wolves: Fans let rip at Kieron Dyer over "ill-informed" Molineux claim Wolves: Nuno Espirito Santo keeping tabs on Liam Moore Wolves: Adama Traore agrees to extend Molineux contract Wolves: Mark Lawrenson hails Wolves ahead of Newcastle encounter Wolves: Juventus withdraw interest for Adama Traore Wolves: Fans hail Leander Dendoncker after Tim Spiers reveals compelling statistic Wolves: Paul Robinson accuses Raul Jimenez of malicious tackle during win at Leeds Wolves: Fans react as Jonny Otto continues his pursuit to full fitness Leeds United: Michael Brown believes Liam Cooper will be risked against Wolves Leeds United: Kevin Phillips discusses defensive issue ahead of Wolves tie Wolves: Nuno Espirito Santo plans to 'evolve' current Molineux squad Wolves: Tony Pulis believes Raul Jimenez is second only to Harry Kane Wolves: Fans have no sympathy for Liverpool over controversial VAR decision Wolves: Patrick Cutrone's loan spell at Fiorentina hampered by 'famous clause'


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Sustainable Water Management in Santorini
Laura Kenny (M.R.P. '17) spent her summer working in historic rainwater harvesting cisterns for an interdisciplinary project on sustainable water management in Santorini, Greece.
With the help of grants from the Atkinson Center for a Sustainable Future and Cornell Institute for European Studies, Kenny traveled to Santorini to work on the project with Professor Gail Holst-Warhaff of the Institute for European Studies and Professor Tammo Steenhuis of the Department for Biological and Environmental Engineering, as well as a group of five students from other departments across Cornell. The project's aim was to study the rainwater harvesting cisterns and undertake an assessment on reusing cisterns for future water storage and/or rainwater collection and to increase awareness of rainwater harvesting potential at the community level.
Historically, buildings on the arid island were constructed with water collection systems and holding cisterns for rainwater that was used for drinking, bathing, cooking, and recreation. This method was sufficient in providing enough water for the island's needs, but, as tourism increased, desalination plants largely replaced the historic rainwater harvesting cisterns, causing many to fall into disrepair.
The research group worked with the Santorini Water Board to examine several cisterns on the island for potential reuse. "The work required us to climb down into the cisterns to inspect for structural issues, learn about the history and community surrounding each cistern, and research historical water use practices," said Kenny.
Using her research, Kenny's final project creates a "Water Walk" to provide the local residents and tourists with an understanding of water use on the island of Santorini, and to encourage more sustainable practices. Her final project includes a list of recommendations for additional water walk sites, an interactive map, and signage for posting at the proposed sites.
By Patti Witten


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Welcome to the page of the Alaska Chapter of the American Society for Landscape Architects, where the Society's mission is to lead, educate, and participate in the careful stewardship, wise planning, and artful design of our cultural and natural environments. The Alaska Chapter advances this mission by supporting the knowledge and practice of landscape architecture as a means to protect and enhance our natural and built environments in Alaska. The Chapter promotes the landscape architectural profession through advocacy, education, communication, and fellowship.
Jobs, Volunteer, Student Scholarshops, Subcommittees
Sponsors and Sponsorship Information
Job and Scholarship Opportunities
Constitution and Bylaws, Agenda and Meeting Minutes, Alaska Plants, and Annual General Reports
Event and Annual Sponsorship Information and Payment
About the current Executive Committee and standing committee responsibilities and leads
Meet the current board and view committee information.


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NHL Rumor Roundup: What's next for Erik Karlsson and the Senators?
Can't get enough NHL trade rumors? Lyle Richardson's weekly Rumor Roundup column serves as a one-stop guide to the latest rumblings around the league.
Senators defenseman Erik Karlsson was the biggest name in the market heading toward Monday's NHL trade deadline. While the deadline passed with the 27-year-old superstar still a Senator, his long-term future in Ottawa remains uncertain.
TSN's Darren Dreger reported the Golden Knights and Lightning were among six teams attempting to acquire the 26-year-old superstar. Sportnet's Elliotte Friedman said Vegas came the closest, offering up "probably about six pieces," including players and draft picks and willingness to take on Bobby Ryan's contract.
Senators general manager Pierre Dorion remains hopeful of re-signing Karlsson, who's slated to become an unrestricted free agent in July 2019. On Tuesday, the Senators captain said he never requested a trade and is open to signing a long-term extension.
MORE: NHL trade deadline winners and losers
However, the Ottawa Sun's Wayne Scanlan is skeptical, noting Dorion's willingness to entertain offers could make a contract extension unlikely. His colleague Ken Warren believes there will be more teams with interest in Karlsson this summer, increasing the possibility of the Sens receiving an irresistible offer.
The Golden Knights could revisit their interest in Karlsson this summer. They still possess depth in draft picks, prospects and young players to make a competitive bid.
Vegas has over $46 million invested in 17 players for 2018-19, with James Neal, David Perron, William Karlsson and Shea Theodore as their notable free agents. Should the Golden Knights re-sign all of them, they'll still have sufficient cap space to add Karlsson's current $6.5 million salary cap hit for next season and perhaps enough to offer him a lucrative long-term extension.
The Lightning also inquired about Karlsson. However, their acquisition of defenseman Ryan McDonagh from the Rangers takes them out of this summer's potential sweepstakes for the Senators superstar.
Karlsson probably won't be the only player the Senators get inquiries about this summer. Bruce Garrioch reported the Jets and Sharks were among several Western Conference teams interested in winger Zack Smith, while the Blues pursued Mike Hoffman since midseason.
MORE: Grading every trade made on 2018 deadline day
Does Evander Kane have a future in San Jose?
The Sharks landed one of the notable trade candidates before the deadline, acquiring Evander Kane from the Sabres. The 26-year-old left wing wasted little time making an immediate positive impression with his new team, collecting two assists in the Sharks' 5-2 win over Edmonton on Tuesday.
Kane is due to become an unrestricted free agent on July 1, but his tenure in San Jose could extend beyond this season. Sportsnet's Elliotte Friedman noted the Sharks carry over $20 million in salary cap space for 2018-19. That's more than enough to re-sign the winger if he plays well over the remainder of this season.
While Kane has a history of questionable off-ice behavior, he's shown more maturity over the last two years. He could also also benefit from playing alongside respected Sharks stars such as Joe Pavelski, Brent Burns and Joe Thornton once he returns from injury.
As this is Kane's contract year, it's in his best interest to play well if he hopes to secure a lucrative new deal this summer.
GRADES: Jason Botterill flunks first trade deadline test
Blues could be busy this summer
The most startling deal of this year's trade deadline saw the Blues ship veteran center Paul Stastny to the Jets. Stastny, 32, didn't appear in any media trade chatter leading up to deadline day, making move all the more surprising.
Blues GM Doug Armstrong seems to be giving up on the season. Thanks in part to a lack of scoring depth, his club has tumbled out of a playoff spot. By moving out one of the Blues' best playmakers without bringing in a replacement, he failed to address their offensive woes.
It wasn't for lack of effort on Armstrong's part. The Athletic's Jeremy Rutherford reported the Blues GM had talks with the Senators regarding winger Mike Hoffman and the Canadiens about left wing Max Pacioretty. Armstrong, however, was unwilling to part with a top young prospect such as Klim Kostin, Jordan Kyrou, Tage Thompson or Robert Thomas.
How the Blues finish this season could determine Armstrong's course of action this summer. Should they fail to reach the playoffs, perhaps he'll shake up his roster by revisiting his interest in Hoffman or Pacioretty.


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BARGAIN MODERN STYLE VILLA - Detached villa in Torreblanca, totally renovated with panoramic views, 3 bedrooms and 3 bathrooms. As entering you will find 2 bedrooms, 1 having en suite. Both rooms has terraces facing South West. On this floor you will also find a storage/washing room. Marble floors and double glazed windows in whole house. Upstairs you have 1 bedroom en suite. With nice roof terrace, panoramic views and sun all day long. All bedrooms have built in wardrobes. Down stairs you will find a nice open planed kitchen with place for dinning table and living room . Storage will be found here as well and a guest toilet.
From the living room and dining area you have entrance out to barbecue area, dinning area and private pool with shower. The outdoor dining area can be used as covered terrace all year around.
This house is a fantastic holiday home, easy to maintain. With the house comes another terrace/out door space with plenty of storage place. As well private parking belongs to the house.
The area around the house is Zona Verde, restricted area where nothing can be built. 3 minutes drive to Los Pacos, close to train station of Torreblanca and easy access to high way.
As well close to schools and shops, restaurants within walking distance.
Avenida de Las Gaviotas 51
Fuengirola Center
29640 Fuengirola


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A Sweet Salve For Shingles?
A preliminary study done in partnership with the University of Colorado-Denver suggests honey might be effective in treating a viral disease—shingles.
Growing up in Pakistan, Aamir Shahzad of the University of Vienna remembers a close family member coming down with shingles. Later, as a physician in rural parts of the country, he encountered it frequently.
"I noticed many immunocompromised and elderly people suffer with shingles," wrote Shahzad in an email.
The painful rash that characterizes shingles, or herpes zoster, is caused by reactivation of cells infected with Varicella zoster virus, the virus responsible for chickenpox.
The disease often develops into blisters and can cause neurological complications and even death in rare cases.
In Pakistan and many developing nations, the standard antiviral drugs either aren't available or are too expensive, and few get the vaccine.
Shahzad wanted to find a cheaper alternative, so he sought out an expert on shingles, Randall Cohrs, a professor of neurology at University of Colorado-Denver. Cohrs invited Shahzad to come to Denver and work in his lab to test the ability of honey to fight the virus.
When Shahzad grew cells infected with virus in the presence of either clover or Manuka honey, he saw a reduction in the amount of virus. The results are preliminary, but Shahzad suspects honey may have previously undocumented antiviral properties.
"As honey contains various substances which might act synergistically or might contain an unknown active antiviral agent, we are planning to check the difference in antiviral activity of honey from different geographical locations and ultimately to find out honey's exact mechanism of action," wrote Shahzad.
Several hurdles remain, including clinical and safety tests. Notably, the highest concentrations of honey Shahzad tested also proved toxic to cells.
Shahzad cautions that honey should never replace more effective antivirals in the United States, but in places like his home country of Pakistan, it may prove to be a salve for more than a sore throat.


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Nothing says summer like a fruity, refreshing Sangria! And since alcohol can increase the impact of THC by up to 50%. Along with the combination of the terpenes in both the grapes used to make the wine and the cannabinoids in your finest ganja taste delicious! This cannabis infused sangria recipe is sure to be a crowd pleaser. So lets get started:
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This is a simple to follow recipe and you're only going to need a few things.
- 1/8 grams of your finest weed (You could use more if you want. I came up with an 1/8 because I figured I can smoke an 1/8 by myself and drink a bottle of wine by myself so it seemed to be the right amount for me personally)
- 1 bottle of red wine (I used a Cabernet Sauvignon, my favorite, which ever wine you choose make sure it's a dry red, not a sweet red)
- 10 pineapple chunks about a 1/2inch by a 1/2 inch cubed
- 1 orange sliced up thin
- 10 strawberries hulled and diced
- 14-20 green grapes cut in half
- 1 large pot or saucepan
- Cheesecloth and/or strainer (I use both)
- A pitcher
- Ice
Once you've completed your prep the next part is called fortifying/infusing the wine. In theory Sangria is a fruit infused wine. But we are taking one step further by adding herb to it. Similar to what they have done since biblical times in making mulled wine. In case you don't know the difference between mulled wine and sangria is that you serve sangria cold, that's it.
- Pour the wine into the pot and place it on the stove.
- Add cannabis and half the above listed fruit, of coarse you can use different fruit if you'd like. However this recipe will bring out and enhance several elements of both the wine and the cannabis. It is a fact that vitamin C and citrus add to the entourage effect.
- Boil the mixture at a low flame for about two hours, checking frequently to make sure it's not all evaporating. The process of boiling everything together will decarboxylate the cannabis.
- Strain the wine from the fruit and cannabis through a cheesecloth and/or strainer. See I put the cheesecloth in the strainer but you really don't have to use both.
- Let the wine cool off.
- Fill a pitcher with remaining fruit and ice.
- And finally pour the infused wine you just created into your fruit and ice filled pitcher.
Warning: The results may be stronger than you expected, so take it slow and of coarse enjoy!
FYI: Don't forget to eat the fruit at the the end either. Little fact about me, I got drunk for the 1st time when I was around 4 or 5 years old at a New Years eve party because my very young mother didn't know that the fruit absorbs the liquor and I was eating everybody's cocktail cherries in their drinks. So eat the fruit it's an edible on its own. (I still love cocktail cherries by the way)
Hope you liked my cannabis infused sangria recipe and as always stay lit!