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For the construction of genomic libraries, ∼30 μg of high molecular weight genomic DNA were partially digested with Sau 3AI and electrophoresed on a 0.4% agarose gel. Fragments in the size range 15 to 23 kb were eluted from the gel, ligated with LambdaGEM-11 Bam HI arms, and packaged in vitro using Packagene extracts (Promega).
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The insertions at the 2j inversion breakpoints do not show homology at the nucleotide level to any known sequence in the databases and thus appear to correspond to a previously undescribed TE. We have named this TE after the Italian astronomer, mathematician, and physicist Galileo Galilei (1564-1642).
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note
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A 5-kb probe of the λj3.1 proximal end that contains most of the BD insertion was in situ hybridized to the polytene chromosomes of seven D. buizatii lines. The total number of euchromatic signals found per genome was 55 in st-1, 44 in st-3, 47 in st-4, 34 in j-1, 34 in j-8, 37 in j-11, and 28 in j-13 (23). On average, 11.8% of each line's signals were exclusive.
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The PCR products were purified from 1% agarose gels, reamplified with one of the sequencing primers shown in Fig. 2, and sequenced in an ALFexpress DNA automated sequencer (Pharmacia Biotech). Both strands of each fragment were sequenced completely using primers separated by 323 to 466 nucleotides.
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43
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Two sets of primers were used to amplify simultaneously a 271-bp 5′ fragment of the Pp1α-96A cDNA and 438 bp of the Gapdh cDNA (internal control) from the total cDNA. In both cases the primer sites were selected to span introns (of 61 bp and 69 bp, respectively). Gapdh primers were designed on the basis of the sequence of D. hydei [K. M. Wojtas, L. von Kalm, J. R. Weaver, D. T. Sullivan, Genetics 132, 789 (1992)]. PCRs were performed with varying amounts of the reverse transcription reaction product as template and different concentrations of the two sets of primers.
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The Northern blot hybridization was done with ∼30 μg of total RNA from embryos, larvae, pupae, and adults of the st-1 and j-1 lines as described [A.-K. Rost, in Quantitation of mRNA by Polymerase Chain Reaction: Nonradioactive PCR Methods. Th. Köhler et al., Eds. (Springer-Verlag, Berlin, 1995), pp. 93-114].
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Supported by a doctoral fellowship and a travel grant from the Comissionat per a Universitats i Recerca (Generalitat de Catalunya, Spain) (M.C.) and grant PB95-0607 from the Dirección General de Investigación Cientifica y Técnica (Ministerio de Educación y Cultura, Spain) (A.R.). We thank J. Zhang for his help in the RNA work; J. Barbé, O. Cabré, and A. Tapias for material and technical help; B. Schmitt for the nAcRβ-96A clone; J. S. F. Barker, E. Betrén, A. Fontdevila, and F. M. Sene for D. buzzatii stocks; and M. Aguadé, M. Ashburner, A. Berry, F. Brunet, B. Charlesworth, and R. de Frutos for valuable comments and helpful discussions.
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