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First published online January 18, 2008
doi: 10.1242/10.1242/dev.00543

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An endogenous Su(Hw) insulator separates the yellow gene from the Achaete-scute gene complex in Drosophila

Anton Golovnin^1,2,3,, Inna Biryukova^1,*,, Olga Romanova^1,, Margarita Silicheva¹, Akeksander Parshikov¹, Ekaterina Savitskaya^1,2, Vincenzo Pirrotta³ and Pavel Georgiev^1,

¹ Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences, Moscow 117334, Russia
² Center for Medical Studies, University of Oslo at the Institute of Gene Biology, Russian Academy of Sciences, Moscow 117334, Russia
³ Department of Zoology, University of Geneva, CH1211 Geneva, Switzerland
^* Present address: Institute de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, 67404 IIIkirch Cedex, France
These authors contributed equally to the work

View larger version (20K): [in this window] [in a new window]   Fig. 1. The nature and properties of original mutations in AS-C. (A) Schematic presentation of the yellow/ac/sc region. Small arrowheads show insertions of the P elements associated with certain mutations. Thick horizontal arrows show the direction of transcription of the yellow, ac and sc genes. The arrows in boxes indicate the orientation of the P elements. The structure of the scls1, scls2 and scls3 alleles was described previously (Golovnin et al., 1999). (B) Phenotypes of the indicated sc bristle mutations in males. The standard nomenclature for each bristle is indicated as follows (Lindsley and Zimm, 1992): HU, humeral; AOR, anterior orbital; PS, presutural; ASA, anterior supra-alar; OC, ocellar; PV, postvertical; ANP, anterior notopleural; SC, scutellar. Only the bristles affected in sc mutations are shown. Empty boxes indicate that the corresponding bristles are present (wild-type phenotype). In all but the scutellar, one quarter black, half black and fully black boxes mean that the corresponding bristle(s) was (were) absent in over 10%, 50% or 90% of the flies, respectively. For scutellars, quarter black, half black and fully black boxes mean that 3-4, 2-3 or 0-1 scutellar bristles, respectively, were present. Number of bristles is the mean of about 100 scored flies. The phenotypes of scls1, scls2 and scls3 flies were taken from Golovnin et al. (Golovnin et al., 1999). The su(Hw)v/su(Hw)f and su(Hw)v/su(Hw)2 transheterozygous lines had similar effect on the scms1 and scms2 mutations.

View larger version (40K): [in this window] [in a new window]   Fig. 2. Binding of in vitro synthesized Su(Hw) to two putative Su(Hw) binding sites in the 125 bp DNA fragment. (A) The sequence of the 125 bp DNA fragment is shown. Putative Su(Hw) binding sites are boxed. The primers used to obtain the DNA fragments are shown as arrows. The mutated residues are indicated below the sequence. The consensus for the Su(Hw) binding site was taken from Scott and Geyer (Scott and Geyer, 1999). (B) Electrophoretic mobility shift assays. The radioactively labeled gypsy, 125 bp fragment, Su(Hw)#1, Su(Hw)#2, Su(Hw)#1*, Su(Hw)#1**, 454 bp and 454* bp fragments were used as probes, incubated with in vitro-synthesized Su(Hw) protein and run on a 1.5% agarose gel (Materials and Methods). One shifted band (indicated by arrows) presumably corresponds to a protein-DNA complex formed by Su(Hw) with only one Su(Hw) binding site. The binding of Su(Hw) to the 125 bp DNA fragment was examined in the presence of competitors. The binding is competed by excess unlabeled Su(Hw)#1* fragment but not by the Su(Hw)#1** fragment with the mutated Su(Hw) binding site.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Study of transgenic lines to test the enhancer-blocking activity. (A) Transposon constructs. The maps of the constructs (not to scale) show the yellow wing (En-w) and body enhancers (En-b) as partially overlapping white boxes and the eye enhancer (Eye) as a white oval. Downward pointing arrows labeled FRT or Lox mark the target sites of the Flp or Cre recombinase, respectively. The 125 bp, 454 bp, 454* bp, 125 bpx3 and 2 kb DNA fragments were inserted at -893 bp relative to the yellow transcription start site. The Su(Hw) insulator was inserted between yellow and white. The yellow and white genes are shown with arrows indicating the direction of transcription. (B) Analysis of yellow and white expression in males from transgenic lines heterozygous for the construct. Small symbols in the boxes indicate the number of independent transgenic lines displaying similar abdominal (black square) or eye (black circle) pigmentation. To determine the y and w phenotypes, the extent of pigmentation in the abdominal cuticle (reflecting the activity of the En-b enhancer) as well as the eye pigmentation of adult flies were estimated visually in 3- to 5-day-old males developing at 25°C (see Materials and Methods). Expression levels were determined without excision of functional elements in the wild type and after excision of the Su(Hw) insulator (Su), of the eye enhancer (E), or the tested DNA fragment (Fr). Abbreviation: su(Hw)-, su(Hw)v/su(Hw)f.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Role of Su(Hw) and Mod(mdg4) in the regulation of ASC. (A) Schematic presentation of the yellow/ac/sc region in the previously described y, ac and sc mutants (Campuzano et al., 1985). The coordinates of the ASC region are as defined in Campuzano et al. Vertical arrows indicate the positions of chromosomal breakpoints associated with the y3P, scv2 and sc8 mutations. The localization of the sc2 and sc5 deletions is indicated by an elongated open box (Campuzano et al., 1985). Arrows with a triangle show insertions of P elements associated with duplication of the yellow sequences. Relative orientations of P elements are indicated by arrows in boxes. Thick horizontal white arrows show the positions and direction of yellow and ASC genes transcripts. The gray oval indicates the putative Su(Hw) binding sites in the 125 bp DNA fragment. (B) The effect of the su(Hw) (su(Hw)v/su(Hw)f and su(Hw)v/su(Hw)2) and mod(mdg4) (mod(mdg4)u1/mod(mdg4)u1 and mod(mdg4)u1/Df(3R)GC14) mutations on the phenotype of the mutations in ASC. The su(Hw)v/su(Hw)f and su(Hw)v/su(Hw)2 transheterozygous lines had similar effects on the mutations in ASC. Phenotypes of the indicated sc mutations were examined in males. The standard nomenclature for bristles whose formation is regulated by ac are as follows (Lindsley and Zimm, 1992): ADC, anterior dorsocentral; PDC, posterior dorsocentral; PSA, posterior supraalar; AVT, anterior vertical; MC, the rows of microchaetae on the notum. Other designations as in Fig. 1. Only affected bristles in ac and sc mutations are shown. Bristle pigmentation: w-v, weak variegation indicates that 1-3 bristles in thorax and head are yellow; m-v, mild variegation shows that about half of bristles are yellow; +, wild-type pigmentation of all bristles. Viability: +, about normal viability; -, lethal. The figures indicate viability for combination of mod(mdg4)u1/mod(mdg4)u1 with sc2, i.e. ratio of sc2 males to yw males obtained in the progeny of heterozygous yw/sc2; mod(mdg4)u1/mod(mdg4)u1 females. The total number of sc2 and yw males scored is shown in brackets.