* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download DIS (1999) 82, 94-95 - Institut de Génétique Humaine
Long non-coding RNA wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Gene nomenclature wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Minimal genome wikipedia , lookup
Human genome wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Ridge (biology) wikipedia , lookup
Pathogenomics wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
X-inactivation wikipedia , lookup
Primary transcript wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Neocentromere wikipedia , lookup
Metagenomics wikipedia , lookup
Genome evolution wikipedia , lookup
Point mutation wikipedia , lookup
Genomic imprinting wikipedia , lookup
History of genetic engineering wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Non-coding DNA wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Genomic library wikipedia , lookup
Microevolution wikipedia , lookup
Genome (book) wikipedia , lookup
Gene expression profiling wikipedia , lookup
Gene expression programming wikipedia , lookup
Gene desert wikipedia , lookup
Designer baby wikipedia , lookup
Transposable element wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
DIS (1999) 82, 94-95 A Drosophila enhancer detector transposon marked with the yellow gene. Isabelle Busseau* and Alain Bucheton Institut de Génétique Humaine, CNRS, 141 rue de la Cardonille, 34396 Montpellier cedex 05, FRANCE *Corresponding author. Phone: 33 (0) 4 99 61 99 48, Fax: 33 (0) 4 99 61 99 01, E-mail: [email protected] Key words: P-mediated transposition; enhancer trap; yellow gene. The P[lyB] enhancer trap vector (figure 1A) was designed to be used in P-element-mediated mutagenesis and enhancer detection in Drosophila melanogaster. It contains the ß-galactosidase reporter gene and the yellow gene as a transformation and transposition marker. It was derived from P[lAwB] (Flybase ID = FBmc0000173), designed to allow rapid cloning and deletion analysis of genomic sequences into which it inserts, and therefore has retained the same properties. Construction of the P[lyB] vector and establishment of transgenic lines P[lyB] was derived from P[lAwB] by replacing the ADH and white sequences from this vector with yellow sequences.The source of the yellow sequences was plasmid Dint (Geyer and Corces, 1987) that contains all DNA sequences from the yellow gene except the intron. The SalI fragment from Dint containing the yellow sequences was first subcloned into the pBluescript KS- vector, in order to place the yellow gene between a XhoI and a XbaI sites. Then the XhoI-XbaI fragment from this clone was ligated to XhoI- and XbaI-cut P[lAwB]. P[lyB] was injected into embryos from the JA strain (carrying both yellow and white mutations) using standard procedures described by Spradling and Rubin (1982) except that puchsπ∆23 (Flybase ID: FBmc0002087) was co-injected as the source of transposase. Several independant transgenic lines were established. Two of them, designated J49 and J92, were characterized further by estimating the rates of transposition and excision of P[lyB]. They both carry the P[lyB] transgene on the second chromosome. Efficient mobilization of P[lyB] Mobilization of P[lyB] was carried out using P[ry+∆2-3](99B) as a stable genomic transposase source, basically using the “jumpstart” scheme of Bellen et al. (1989) modified as shown in figure 1B. Virgin females from J49 or J92 transgenic line were mated to males homozygous for P[ry+∆2-3](99B) and carrying a Cy balancer second chromosome (figure 2). In the progeny of these crosses, [Cy] males, that carry both P[lyB] and P[ry+∆2-3](99B), were selected and mated with virgin females from the JA stock. The occurrence of [y+;Cy] individuals in their progeny reflected transposition events to new chromosomal location. From these experiments, estimations of transposition frequencies of P[lyB] were 4.7% when using line J49 and 3.3% when using line J92. Although these estimations are rough, because very few flies were scored in these experiments, they indicate that the rate of mobilization of P[lyB] is in the usual range (1-5%) for defective P elements mobilized by P[ry+∆2-3](99B) (ENGELS, 1989). Therefore the P[lyB] element can be used efficiently 1 HindIII HindIII EcoRI EcoRI BamHI HindIII EcoRI A BamHI HindIII as an alternative to other enhancer trap elements in cases where the use of a yellow marker appears convenient. B y w J49 y w or J92 JA Line y w y w + + + + [y;Cy] [y+;Cy+] P-lyB P-lyB + + yellow BT KS - + Cy P∆2-3 x Y Pm P∆2-3 y w P-lyB + x Y Cy P∆2-3 [y;Cy+] [y+;Cy] Total % excision % transposition J49 307 31 298 15 651 90.58% 4.66% J92 1307 558 984 44 2893 63.81% 3.26% Excision Transposition No mobilization P BamHI lac Z EcoRI BamHI HindIII EcoRI XbaI BamHI EcoRI HindIII P XhoI P[lyB] Figure 1: A: Sequence organization and partial restriction map of p-lyB. Thick line represent sequences from pBluescript KS-, black boxes represent P element sequences, shaded box represent LacZ sequences, striped box represent yellow sequences. Arrows indicate senses of transcription of LacZ and yellow genes. B: scheme of crosses to estimate the rate of mobilization of p-lyB by P[ry+∆2-3](99B) and results. Acknowledgements We are very grateful to Dr Walter Gehring for the gift of plasmid P[lAwB] and to Nathalie Rémillieux who participated in these experiments when she was a second year student at the Université Paris XI (Orsay). Lines J49 and J92 will be available at the Umea Drosophila Stock Center and at the Bloomington Stock Center. References Bellen, H.J., O'Kane, C.J., Wilson, C., Grossniklaus, U., Pearson, R.K., Gehring, W.J., 1989. P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev. 3, 1288-1300. Geyer P.M., Corces V.G., 1987. Separate regulatory elements responsible for the complex pattern of tissue-specific and developmental transcription of the yellow locus in Drosophila melanogaster. Genes Dev. 1, 996-1004. Robertson, H.M., Preston, C.R., Phillis, R.W., Johnson-Schiltz, D.M., Benz., W.K., Engels, W.R., 1988. A stable source of P-element transposase in Drosophila melanogaster. Genetics 118, 461-470. Spradling, A. and Rubin, G.M., 1982. Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218, 341-347. Wilson, C., Pearson, R.K., Bellen, H.J., O'Kane, C.J., Grossniklaus, U., Gehring, W.J., 1989. P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila. Genes Dev. 3, 1301-1313. 2