Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells.
TL;DR: Copper and cadmium, when added to the growth medium of Drosophila Schneider's line 2 cells, can produce a 30-100 fold induction of metallothionein mRNA levels, and a chimeric gene containing the metallothsionein promoter shows a similar induction when transformed into the cells.
Abstract: The promoter from the metallothionein gene may be a useful conditional promoter for the construction of chimeric genes to be expressed in Drosophila cells in culture. To explore this possibility the responses of the endogenous metallothionein gene and an in vitro constructed chimeric gene containing the metallothionein promoter were examined. Copper and cadmium, when added to the growth medium of Drosophila Schneider's line 2 cells, can produce a 30-100 fold induction of metallothionein mRNA levels. The level of induction depends on the amount of copper or cadmium added to the medium and these mRNA levels remain high for at least four days. Copper is less toxic than cadmium and does not induce a typical heat-shock response in the cells. Finally, a chimeric gene containing the metallothionein promoter shows a similar induction when transformed into the cells.
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TL;DR: Evidence that animal circRNAs are generated cotranscriptionally and that their production rate is mainly determined by intronic sequences is provided and it is demonstrated that circularization and splicing compete against each other.
2,225 citations
Cites background from "Characterization and use of the Dro..."
...Transcription in all these minigenes is under the control of the metallothionein (MT) promoter, which is copper inducible (Bunch et al., 1988)....
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TL;DR: The characterization of three mammalian Slit homologs are described and it is shown that the Drosophila Slit protein and at least one of the mammals Slit proteins, Slit2, are proteolytically processed and show specific, high-affinity binding to Robo proteins.
1,167 citations
Cites background from "Characterization and use of the Dro..."
...…dSlit in Drosophila S2 cells, fragments containing the axons in culture, either when hSlit2 was presented as a Drosophila slit ORF and the human alkaline phosphatase gene were point source or in a uniform concentration (unpublished cloned into the S2 transfection vector pRmHa3 (Bunch et al., 1988)....
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TL;DR: Drosophila melanogaster Tsc2 seems to be the critical target of Akt in mediating growth signals for the insulin signalling pathway, and Stimulating Akt/PKB signalling in vivo markedly increases cell growth/size, disrupts the Tsc1–Tsc2 complex and disturbs the distinct subcellular localization of T Sc2.
Abstract: The direct mechanism by which the serine/threonine kinase Akt (also known as protein kinase B (PKB)) regulates cell growth is unknown. Here, we report that Drosophila melanogaster Akt/PKB stimulates growth by phosphorylating the tuberous sclerosis complex 2 (Tsc2) tumour suppressor and inhibiting formation of a Tsc1-Tsc2 complex. We show that Akt/PKB directly phosphorylates Drosophila Tsc2 in vitro at the conserved residues, Ser 924 and Thr 1518. Mutation of these sites renders Tsc2 insensitive to Akt/PKB signalling, increasing the stability of the Tsc1-Tsc2 complex within the cell. Stimulating Akt/PKB signalling in vivo markedly increases cell growth/size, disrupts the Tsc1-Tsc2 complex and disturbs the distinct subcellular localization of Tsc1 and Tsc2. Furthermore, all Akt/PKB growth signals are blocked by expression of a Tsc2 mutant lacking Akt phosphorylation sites. Thus, Tsc2 seems to be the critical target of Akt in mediating growth signals for the insulin signalling pathway.
963 citations
TL;DR: The steroid hormone ecdysone triggers coordinate changes in Drosophila tissue development that result in metamorphosis and a gene, EcR, is isolated and characterized for a new steroid receptor homolog and it is shown that it encodes an ecDysone receptor.
943 citations
TL;DR: It is suggested that Notch may act as a multifunctional receptor whose 36 EGF repeats form a tandem array of discrete ligand-binding units, each of which may potentially interact with several different proteins during development.
855 citations
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TL;DR: New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors and mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences.
14,954 citations
TL;DR: Labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.
10,489 citations
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TL;DR: The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.
Abstract: Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.
7,858 citations
TL;DR: A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity.
Abstract: A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently.
7,457 citations
3,603 citations