Toward Anopheles Transformation: Minos Element Activity in Anopheline Cells and Embryos

Corrections should read UOP ADS-34; and (ii)inImpacts of Molecular Sieves COLLOQUIUM PAPER. For the article ‘‘Synthetic zeolites and other on Human Welfare, the phrase, ‘‘From these numbers,’’ should be microporous oxide molecular sieves’’ by John D. Sherman, which deleted. appeared in number 7, March 30, 1999, of Proc. Natl. Acad. Sci. Correction published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞ USA (96, 3471–3478), the author notes the following corrections: pnas.110133597. Text and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞ (i)inLinear Paraffins for Biodegradable Detergents, OP ADS-34 pnas.110133597 CELL BIOLOGY. For the article ‘‘Tyrosine phosphorylation of p62dok GENETICS. In the article ‘‘Toward Anopheles transformation: by p210bcr-abl inhibits RasGAP activity’’ by Nobuhiro Kashige, Minos element activity in anopheline cells and embryos’’ by Nick Carpino, and Ryuji Kobayashi, which appeared in number Flaminia Catteruccia, Tony Nolan, Claudia Blass, Hans-Michael 5, February 29, 2000, of Proc. Natl. Acad. Sci. USA (97, 2093– Mu¨ller, Andrea Crisanti, Fotis C. Kafatos, and Thanasis G. 2098), the authors note that the image in lane 12 of Fig. 6a was Loukeris, which appeared in number 5, February 29, 2000, of mistakenly deleted in the printing process. The complete figure Proc. Natl. Acad. Sci. USA (97, 2157–2162), the authors note that and its legend are shown below. three mistakes were introduced inadvertently in assembling Fig. Correction published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞ pnas.110137997. Text and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞ 4. The revised Fig. 4 printed below includes the correct photo- pnas.110137997 graph of E7 insertion at 21E, as well as the correct chromosomal locations of E4 at 25D (2L) and E5 at 36B (3R). Fig. 6. Peptide competition analysis indicates that Tyr-296 and Tyr-315 play a critical role in the binding of p62dok to RasGAP. The ability of the diphosphopeptide to inhibit binding of Dok-1 to GAP suggests that the proper positioning of pTyr-296 and pTyr-315 in tandem is critical for the interaction of the two molecules. (a) Combinations of phosphopeptides corresponding to the regions surrounding individual tyrosines in p62dok fail to inhibit the binding of p62dok to RasGAP. However, a diphosphopeptide corresponding to residues 293–322 is able to inhibit binding of Dok-1 to the GAP SH2-SH3-SH2 region. Binding analysis was conducted as described. Synthetic phosphopeptides used for this experiment were: 1, SPPALpYAEPLDS (pTyr-296); 2, SQD- SLpYSDPLDS (pTyr-315); 3, PKEDPIpYDEPEGL (pTyr-362); 4, VPPQG LpYDL- PREPK (pTyr-377); 5, RVKEEGpYELPYNPATDD (pTyr-398); 6, NPATDD pYAVP- PPR (pTyr-409); and diphosphopeptide, PALpYAEPLDSLRIAPCPSQDS LpYSDPLDST (pTyr-296 and pTyr-315). For control, unphosphorylated pep- Fig. 4. (A) Sequences of the Minos insertion sites in the genome of Sua 5.1* tides were used. Each phosphopeptide was added in concentration of 50 ␮M. and Sua 4.0 cells. Chromosomal flanking sequences are represented with (b) Dose-dependent inhibition of p62dok binding to RasGAP by diphosphopep- capital letters in italics. Small lettering represents the sequences of the Minos tide (Dok-1 aa 293–322). The diphosphopeptide was added in concentration end. The expected TA dinucleotide of the insertion site is shown in bold. The of 0.5, 5, or 50 ␮M. The unphosphorylated peptide was added in concentration chromosomal divisions and subdivisions from which the flanking sequences of 50 ␮M. (c) Dose-dependent inhibition of a truncated Dok-1 (the truncation were derived are indicated with the chromosomal arm listed in parenthesis. construct 316: residues 316–481) binding to RasGAP by diphosphopeptide. (B) Typical results of determining the location of origin of the rescued genomic The diphosphopeptide was added in concentration of 0.5, 5, or 50 ␮M. The fragments by in situ localization to polytene chromosomes of the Suakoko unphosphorylated peptide was added in concentration of 50 ␮M. mosquito strain. 6236 ͉ PNAS ͉ May 23, 2000 ͉ vol. 97 ͉ no. 11 Downloaded by guest on September 24, 2021 PLANT BIOLOGY. For the article ‘‘Oryza sativa PSK gene encodes a precursor of phytosulfokine-␣, a sulfated peptide growth factor found in plants’’ by Heping Yang, Yoshikatsu Matsubayashi, Kenzo Nakamura, and Youji Sakagami, which appeared in number 23, November 9, 1999, of Proc. Natl. Acad. Sci. USA (96, 13560–13565), the authors note the following correction. In line 18 of the first column on page 13565, ‘‘ϩ5 positions’’ should read ‘‘ϩ3 positions.’’ Correction published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞ pnas.120161097. Text and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞ pnas.120161097 CORRECTIONS PNAS ͉ May 23, 2000 ͉ vol. 97 ͉ no. 11 ͉ 6237 Downloaded by guest on September 24, 2021 Toward Anopheles transformation: Minos element activity in anopheline cells and embryos Flaminia Catteruccia†, Tony Nolan†, Claudia Blass‡, Hans-Michael Mu¨ ller‡, Andrea Crisanti†, Fotis C. Kafatos‡, and Thanasis G. Loukeris‡§ ‡European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; and †Imperial College of Science, Technology, and Medicine, Imperial College Road, London SW7 2AZ, United Kingdom Contributed by Fotis C. Kafatos, December 23, 1999 The ability of the Minos transposable element to function as a other than Drosophila (2). We used as a marker GFP and the transformation vector in anopheline mosquitoes was assessed. hygromycin B phosphotransferase gene under the control of the Two recently established Anopheles gambiae cell lines were stably Drosophila Hsp70 promoter. We first demonstrated that Minos transformed by using marked Minos transposons in the presence integrates in a transposase-dependent manner in the chromo- of a helper plasmid expressing transposase. The markers were somes of newly established cell lines of A. gambiae. We then either the green fluorescent protein or the hygromycin B phos- confirmed that the element is also mobile in anopheline em- photransferase gene driven by the Drosophila Hsp70 promoter. bryos, by pilot injection experiments coupled with an interplas- Cloning and sequencing of the integration sites demonstrated that mid transposition assay. insertions in the cell genome occurred through the action of Minos transposase. Furthermore, an interplasmid transposition assay Materials and Methods established that Minos transposase is active in the cytoplasmic Plasmid Construction. The helper plasmid pHSS6hsILMi20 has environment of Anopheles stephensi embryos: interplasmid trans- been described (16). The transformation vector pMinHyg was position events isolated from injected preblastoderm embryos constructed by inserting, between the left and right arms of were identified as Minos transposase-mediated integrations, and Minos, in the pMiNot vector (2) sequentially two fragments. A no events were recorded in the absence of an active transposase. fragment, containing hygromycin B phosphotransferase gene These results demonstrate that Minos vectors are suitable candi- under the control of the Hsp70 promoter from Drosophila dates for germ-line transformation of anopheline mosquitoes. melanogaster, and a DNA cassette, which includes a 2.6-kb fragment of the D. melanogaster act5C promoter and a 0.8-kb erm-line transformation provides an important link be- fragment of the Hsp70 terminator. Gtween gene identification and gene function. This approach The plasmid pLaDHFRR used in the excision assay was is especially necessary for organisms in which genetic analysis is constructed as follows. The left and right inverted repeats (IRs) limited. Characteristic example is anopheline mosquitoes, the of Minos including bases 1–350 and 1480–1777, respectively of obligatory vectors of a devastating infectious disease, malaria. the Minos sequences together with Drosophila hydei flanks were Although intensifying molecular studies in Anopheles are iden- PCR-amplified and subcloned separately. The left IR was trans- tifying new genes at an increasing rate, functional gene charac- ferred within the PstI site of the BluescriptKSII (Stratagene). terization is hindered by limitations of genetic analysis and the The right IR fused to sequences containing a dihydrofolate lack of germ-line transformation technology. reductase (DHFR) gene linked to the proximal promoter of the Efforts to transform anophelines have been pursued since Drosophila actin 5C gene (nucleotides Ϫ122 to ϩ88) was trans- the first and only reported case of foreign DNA introduction ferred into the same plasmid as an EcoRI–SalI fragment. In this into the Anopheles gambiae genome 12 years ago (1). These way a Minos transposon carrying internally (between the two efforts were intensified after the successful development of IRs) the BluescriptKSII sequences and the actin DHFR gene was routine transformation techniques using the Minos transpos- generated. Externally, a PstI–EcoRI fragment containing the able element in the Mediterranean fruitfly Ceratitis capitata tetracycline-resistance gene was introduced as spacer between (2) and the mariner and Hermes elements in the yellow fever mosquito Aedes aegypti (3, 4). the two IRs. Since then, both transgenesis and transposon mobility assays The plasmid pLHGR used to stably transform Sua 5.1* cells have been used to establish that several transposable elements derived from the plasmid pLaDHFRR by replacing the se- are efficient gene transfer vectors with a broad host range (5–14). quences

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