Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.     

Two puff-specific proteins bind within the 2.5 kb upstream region of theDrosophila melanogaster Sgs-4 gene

TheDrosophila nuclear proteins Bj6 and Bx42 characterized previously are detected in a series of developmentally active puffs on salivary gland chromosomes. Here the binding of both proteins at puff 3C11-12 containing the glue protein geneSgs-4 is described in more detail. By deletion analysis we show that both proteins bind within a chromosomal segment containing 17–19 kb of DNA surrounding theSgs-4 gene. They are detectable at this site during the intermoult stages, before the puff regresses in response to the moulting hormone ecdysone. If theSgs-4 gene together with flanking DNA sequences is brought into a different chromosomal position by P element transfer, both proteins are detected at this new location. Both proteins are bound to the chromosome within the range of 2.5 kb DNA upstream of theSgs-4 gene. A strain containing a 52 bp deletion within this region fails to bind Bx42 protein suggesting that the missing DNA, which overlaps a hypersensitive region, may be required for the binding of the Bx42 protein.     

A DNase I hypersensitive site flanks an origin of DNA replication and amplification in Sciara

In chromosomes of metazoa, the assembly of the genome into chromatin makes an important but poorly understood contribution to determining where DNA replication will initiate. We addressed this issue by studying the developmental progression of the location of the DNA replication origin (ORI) and alterations in chromatin structure in one of the best-mapped ORIs in metazoa, that found in DNA puff II/9A of the fly Sciara coprophila. We found that DNA synthesis for both normal chromosomal endoduplication and DNA amplification initiates within the same 5.5 kb EcoRI fragment. We showed that irrespective of the mode of ORI function--replication or amplification--chromatin over the 1 kb major ORI is never remodeled into a conventional DNase I hypersensitive site (DH site). Instead, we found that the major site of alterations to chromatin structure at this locus is a large (approximately 400 bp) DH site located 600 bp away from the major ORI, at a position where the frequency of replication initiation events falls dramatically. We describe a tight positive correlation between ORI activity, strength of this DH site, and the intranuclear titer of protein factor(s) that bind the DH site in a sequence-specific manner. We propose that the Sciara replicator in locus II/9A is composed of sequences that reside within the ORI per se as well as sequences encompassed by the DH site.     

Ribosomal DNA of the fly Sciara coprophila has a very small and homogeneous repeat unit

Summary In this report we show by hybridization of restriction fragments and by Miller spreads that the unit repeat of the fly Sciara coprophila is only 8.4 kb which is the smallest known for a multicellular eukaryote. The 8.4 kb EcoR1 fragment containing a complete unit of Sciara rDNA was cloned in pBR322, and mapped by the method of Parker (1977) and also by double digestion. The coding regions for 28S, 18S, and 5.8S RNA were localized by the method of Berk and Sharp (1977). From these data we conclude that the nontranscribed spacer, external transcribed spacer, and internal transcribed spacer are all shorter than in other organisms, thereby giving rise to the shorter overall rDNA repeat unit of Sciara.At least 90% of the Sciara rDNA repeats are homogeneous, with a length of 8.4 kb, but a 700 bp ladder of minor bands can also be found in digestions of total genome DNA. This profile of major and minor bands is identical between the X and X chromosomes, as seen by a comparison of several genotypes.There are only 45 rRNA genes per X chromosome of Sciara (Gerbi and Crouse, 1976). These can easily be counted by low magnification Miller speads which show that virtually all gene copies are actively being transcribed in the stage of spermatogenesis examined. This is the first demonstration for any reiterated gene family where all copies are shown to be simultaneously active.Present address same as last author     

Cytogenetic analysis of region 2B3-4-2B11 of the X-chromosome of Drosophila melanogaster

About 160 kb of DNA were cloned from the 2B region of the X chromosome, where the early ecdysone puff develops and the ecs locus is located. On the physical map of this sequence the positions of 13 chromosome rearrangement breakpoints interfering with both puff development and the ecs locus proximally and distally, were plotted by means of in situ hybridization. The maximal size of the ecs locus is about 100 kb (between the breakpoint of In(1)Hw ^49c and the proximal end of Df(1)St472) The DNA sequences essential for normal puffing are located within the ecs locus between the In(1)br ^lt103 and Df(1)St472 breakpoints and comprise about 65 kb. Thus the puff develops as a result of ecs activation. Since Df(1)P154, which reduces the puff size and removes the proximal part of the ecs locus, does not prevent puff induction by ecdysone, while removing the distal part of the locus by Df(1)St469 completely stops development of the puff, we conclude that the regulatory zone of the locus, which reacts to hormone is located in the distal parts of both the puff and the locus, proximal to the breakpoint of In(1)br ^lt103 .Since In(1)br ^lt103 , Df(1)pn7b and Df(1)br ^R1 damage ecs but do not prevent puffing it is proposed that there is a second regulatory zone for this locus with a minimal size of 15–20 kb (between the breakpoints of Df(1)br ^R1 and In(1)br ^lt103). After cytogenetic and electron microscopic analysis of 2B puff formation it seems very likely that the site of puff formation is situated in the proximal part of 2B3-4 and after enhancement of ecs expression by hormone it spreads proximally to the 2B6 band which does not puff. When the puff regresses at puff stages (PS)10-11 its material does not condense completely and a zone of residual puffing joins the condensed material located distal to it. This material can give the impression of a separate band, designated 2B5 in Bridges' map. For convenience we propose to call the site giving rise to the puff as 2B3-5.     

DNA transfection in the ecdysteroid-responsive GV1 cell line from the tobacco hornworm, Manduca sexta

Summary The embryonic cell line, GV1, from Manduca sexta was transiently transfected with DNA constructs of the Drosophila hsp70 promoter fused to either a β-galactosidase (pXH70ZT) or a chloramphenicol acetyl transferase (HSP-CAT-1) reporter gene using lipofectin. Optimal cell density, DNA:lipofectin ratio, and time of incubation were varied to determine the optimal conditions: 2 × 10⁵ cells/ml, 1:3, and 5 h. Under these conditions, the transfection efficiency was about 40%. Heat inducibility of two hsp70 constructs was compared. The HSP-CAT-1, containing 1127 bp of upstream sequence, was more sensitive to heat shock than that of pXH70ZT, containing only 194 bp of upstream sequence. Thus, the 1127 bp hsp70 promoter appears to be a better inducible promoter in these cells. A 2 kb fragment of the proximal promoter region of the MHR3 gene containing a putative ecdysone response element was shown to be responsive to 20-hydroxyecdysone after its transfection into these cells.     

Die puff-Musterfolgen der Speicheldrüsenchromosomen aus wanderlarven und vorpuppen von Drosophila melanogaster in vitro

Late larval salivary glands of D. melanogaster of an exactly defined developmental stage (VP 0, i.e. prepupae ot later than 15 min after formation of the puparium) are cultured under sterile conditions in three standard media for insect tissue culture and in Ringer solution. In chromosomes II and III, variations in puff number and size are the same in vivo and in vitro, and almost all changes in puffing pattern are very similar to those appearing in normal development. They are the same in the four media. No additional puff is ever induced due to the medium. By contrast, salivary gland chromosomes from larvae of the late third instar before pupation do show different alterations in vitro than in vivo. This points to a threshold in the course of the puffing pattern between puff stage 8/9 and 10/11. The appearance of a substance causing prepupal changes in puffing is strictly correlated with the formation of the pupanium and the beginning of the intermoult phase in the prepupa. Comparing the results of the experiments it can be stated that the new control system is not based solely on the absence of ecdysone, but also on the existence of another inducer. Immediately after puparium formation the control by ecdysone is still active, together with the control by the supposed inducer. Later, control by ecdysone respectively by the puffs of the ecdysone cycle is substituted by the new control system, up to the next moult. As far as the chemical nature of the puffing inducer in the intermoult phase is concerned, further investigations are necessary.