A novel genetic tool for clonal analysis of fourth chromosome mutations

Abstract

The fourth chromosome of Drosophila remains one of the most intractable regions of the fly genome to genetic analysis. The main difficulty posed to the genetic analyses of mutations on this chromosome arises from the fact that it does not undergo meiotic recombination, which makes recombination mapping impossible, and also prevents clonal analysis of mutations, a technique which relies on recombination to introduce the prerequisite recessive markers and FLP-recombinase recognition targets (FRT). Here we introduce a method that overcomes these limitations and allows for the generation of single Minute haplo-4 clones of any fourth chromosome mutant gene in tissues of developing and adult flies.

The KRÜPPEL-Like Transcription Factor DATILÓGRAFO Is Required in Specific Cholinergic Neurons for Sexual Receptivity in Drosophila Females

“…The single breakpoints of the chromosomal deletions Df(4)C1-7A , Df(4)B6-4A , and Df(4)B6-2D and the compound chromosome C(4)DRA-1 were described previously [21] , [22] . The mutations KG02689 ( dati 1 ) and KG01667 ( dati 2 ) were also described previously [22] , [23] and correspond to single P element insertions in the second intron and 300 bp upstream of the first exon of CG2052 , respectively. dati F11.4 was generated by the excision of the P-element KG02689 inserted in dati 1 and corresponds to a precise excision as revealed by direct sequencing of the region that flanks the insertion.…”

“…UAS- dati F32D is a leaky UAS transgene containing the cDNA of dati (GH06573, CG2052 -PA) that rescues the embryonic lethality of l(4)102CDd 2 up to the first instar in the absence of a driver and its insertion was determined to be on the second chromosome in the neurally expressed gene Mmp2 . The D. melanogaster stocks used were as follows: wild-type Canton-S (CS), w 1118 , y w , y w ; Df(4)B6-4A/ ci D spa p ol , y w ; Df(4)B6-2D/ ci D spa p ol , y w ; Df(4)C1-7A/C(4)DRA-1, y w ; Df(4)BH/C(4)DRA-1, y w ; C(4)DRA-1/ dati 1 , y w ; dati F11.4 , y w ; SM/ Cha -Gal4 UAS-GFP; C(4)DRA-1/ dati 1 , y w hs-FLP; SM/FRT42D Actin -Gal4; ci D spa / dati 1 , y w hs-FLP; SM/FYGal80T; spa pol [23] , w ; UAS-mCherry.NLS , w ; dati RNAi/ dati RNAi [FBst0472372], w UAS-dcr-2 ; dati RNAi, y w hs-FLP ; SM /FRT42D Actin -Gal4; C(4)DRA-1/ dati 1 , elav -Gal4 [FBst0008760], w ; TM3 , Sb / repo -Gal4 [FBst0007415], w ; CyO/ Cha -Gal4 UAS-GFP [FBst0006793], w ; ple -Gal4 [FBst0008848], w ; Ddc -Gal4 [FBst0007009], w ; Gad -Gal4 (gift of T. Sakai) [17] , Ubi-GFP.NLS [FBst0005626], Ubi-RFP.NLS [FBst0035496], and FYT/GAF [23] .…”

“…Clonal analyses were performed using the FYT (FLP-recombinase recognition target site- yellow + -Translocation) system previously described [23] . After clone induction, third instar larvae containing GFP+ clones were handpicked, placed in a single vial, and allowed to develop up to adults 3 to 6 d old.…”

Calcium and egg activation in Drosophila

“…However, a method to generate mitotic clones for fourth chromosome genes in the soma has just been reported [24]. Future tweaks to this system may make it possible to induce loss of CaMKII expression in the germline while maintaining normal expression in the soma.…”

GENETIC ANALYSIS OF MATE DISCRIMINATION INDROSOPHILA SIMULANS

“…More recently, the isolation and characterization of sex determination genes significantly increased our understanding of dimorphic development of the nervous system and female mating choice (Baker et al 2001). Also, a series of Drosophila melanogaster mutants that disrupt female choice for conspecific males has been isolated and new methods for their analysis developed (Nakano et al 2001;Sousa-Neves et al 2005;Juni and Yamamoto 2009;Sousa-Neves and Schinaman 2012).…”