A temperature-sensitive mutant affecting the process of chorion gene amplification in Drosophila melanogaster

K575 is a temperature-sensitive female sterile mutant which shows abnormal chorion structure and subnormal amounts of the major chorion proteins at the restrictive temperature. These phenotypes apparently result from a temperature-sensitive defect in amplification. Both clusters of chorion genes are affected, indicating that the gene operates in trans.     

Coding and potential regulatory sequences of a cluster of chorion genes in Drosophila melanogaster

We have characterized at the nucleotide level a 4.8-kilobase pair segment of the third chromosome of Droophila melanogaster, which contains a cluster of three chorion genes, s 18-1, s 15-1 and s 19-1. These genes are tandemly oriented and share the same basic organization: a small and a large exon separated by a short intron in the signal peptide region. In the coding region, limited similarities at the DNA and protein level suggest a common but distant evolutionary origin. The flanking sequences were searched for elements that might be involved in controlling the tissue-specific and temporally regulated expression and the selective amplification of the chorion genes. A good candidate for a cis-regulatory element is the hexamer, TCACGT, which is found in all three genes in a highly significant position, 23 to 27 nucleotides upstream of the TATA-box, accompanied by additional, less exact similarities. Palindromes and short inverted repeats that are found in the vicinity of their complement are non-uniformly distributed: they are most concentrated in the 3 flanking part of all three genes, in and near regions of unusually high A and T content. The highest number of dyad symmetries, remiiscent of sequences that function as viral replication origins, is found associated with the T- and A-rich regions between genes s18-1 and s15-1.     

The Drosophila ACE3 chorion element autonomously induces amplification.

The Drosophila chorion genes amplify in the follicle cells by repeated rounds of reinitiation of DNA replication. ACE3 (amplification control element from the third chromosome) has been identified by a series of deletion experiments as an important control element for amplification of the third-chromosome chorion cluster. Several elements that quantitatively enhance amplification also have been defined. We show that a single 440-bp ACE3 sequence is sufficient to regulate amplification with proper developmental specificity autonomously from other chorion DNA sequences and regulatory elements. Although ACE3 is sufficient for amplification, the levels of amplification are low even when ACE3 is present in multiple copies. When controlled solely by ACE3, amplification initiates either at ACE3 or within closely linked sequences. Amplification of an ACE3 transposon insertion produces a gradient of amplified DNA that extends into flanking sequences approximately the same distance as does the amplification gradient at the endogenous chorion locus. The profile and extent of the amplified gradient imply that the low levels of amplification observed are the result of limited rounds of initiation of DNA replication. Transposon inserts containing multiple copies of ACE3 in a tandem, head-to-tail array are maintained stably in the chromosome. However, mobilization of the P-element transposons containing ACE3 multimers results in deletions within the array at a high frequency.     

Three-dimensional reconstruction of innermost chorion layer from Drosophila melanogaster

A low-resolution three-dimensional structure of the crystalline innermost chorion layer (ICL) has been calculated from electron microscope images of tilted negatively stained crystals. The isolated ICL is a single layer, about 12 nm thick and appears to be made up of two types of subunits, each approximately 3 nm in diameter, arranged regularly as groups of four heterodimers in space group C222. Linking density between these groups of subunits, maintaining the integrity of the layer, appears to be confined mainly to the outer surfaces of the ICL.     

Three-dimensional reconstruction of innermost chorion layer from Drosophila melanogaster

A low-resolution three-dimensional structure of the crystalline innermost chorion layer (ICL) has been calculated from electron microscope images of tilted negatively stained crystals. The isolated ICL is a single layer, about 12 nm thick and appears to be made up of two types of subunits, each approximately 3 nm in diameter, arranged regularly as groups of four heterodimers in space group C222. Linking density between these groups of subunits, maintaining the integrity of the layer, appears to be confined mainly to the outer surfaces of the ICL.     

Localization of a cis-acting element responsible for the developmentally regulated amplification of Drosophila chorion genes

Late in oogenesis two clusters of Drosophila chorion genes and flanking DNA sequences undergo specific amplification in ovarian follicle cells. Lines were constructed using P-element-mediated transformation in which DNA segments derived from the chorion gene cluster at 66D on chromosome III had been inserted at new chromosomal locations. Only transposons that contained a specific 3.8 kb genomic segment derived from the cluster underwent amplification during oogenesis, which occurred with apparently normal tissue and temporal specificity. Adjacent nonchorion sequences also underwent amplification. However, the ability of a transposon to replicate differentially was subject to position effect. These studies provide evidence for the existence of a specific, cis-acting element controlling chorion gene amplification, which includes an origin for disproportionate DNA replication. Attempts to induce amplification with subfragments of the 3.8 kb segment were unsuccessful, suggesting that much of this fragment may be required for amplification.     

Identification and time of synthesis of chorion proteins in Drosophila melanogaster.

The chorion of Drosophila melanogaster consists of proteins secreted by the follicular epithelium during late oogenesis. Petri, Wyman and Kafatos (1976) have described six major protein components of the Drosophila chorion and reported the synthesis of these proteins in vitro by mass-isolated egg chambers. We have used two-dimensional gel electrophoresis to identify approximately twenty components in highly purified chorion preparations. The synthesis patterns of these proteins in vivo were determined by isolating egg chambers of different developmental stages from flies injected with 14C amino acids. Chorion proteins constitute a large fraction of the protein synthesized by ovarian egg chambers in stages 12--14. The sizes and times of synthesis of the chorion proteins correlate closely with the production of poly(A)-containing RNAs by the follicle cells (Spradling and Mahowald, 1979).     

Transfer RNA genes of Drosophila melanogaster.

Three recombinant plasmids containing randomly sheared genomic D. melanogaster tRNAs have been identified and characterized in detail. One of these, the plasmid 14C4, has a D. melanogaster (Dm) DNA segment of 18 kb, and has three tRNA2Arg and two tRNAAsN genes. The second plasmid, 38B10, has tRNAHis genes, while the third plasmid, 63H5, contains coding sequences for tRNA2Asp. The Dm DNA segments in each recombinant plasmid are derived from unique cytogenetic loci. 14C4 is from 84 F, 38B10 is from 48 F and 63H5 is from 70 A.     

The 5S genes of Drosophila melanogaster.

We have cloned embryonic Drosophila DNA using the poly (dA-DT) connector method (Lobban and Kaiser, 1973) and the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975) as a cloning vehicle. Two clones, containing hybrid plasmids with sequences complementary to a 5S RNA probe isolated from Drosophila tissue culture cells, were identified by the Grunstein and Hogness (1975) colony hybridization procedure. One hybrid plasmid has a Drosophila insert which is comprised solely of tandem repeats of the 5S gene plus spacer sequences. The other plasmid contains an insert which has about 20 tandem 5S repeat units plus an additional 4 kilobases of adjacent sequences. The size of the 5S repeat unit was determined by gel electrophoresis and was found to be approximately 375 base pairs. We present a restriction map of both plasmids, and a detailed map of of the5S repeat unit. The 5S repat unit shows slight length and sequence heterogeneity. We present evidence suggesting that the 5S genes in Drosophila melanogaster may be arranged in a single continuous cluster.     

Multiple replication origins are used during Drosophila chorion gene amplification

DNA from Drosophila egg chambers undergoing chorion gene amplification was analyzed using the two-dimensional gel technique of Brewer and Fangman. At stage 10, 34% of DNA molecules from the maximally amplified region of the third chromosome chorion gene cluster contained replication forks or bubbles. These nonlinear forms were intermediates in the process of amplification; they were confined to follicle cells, and were found only within the replicating region during the time of amplification. Multiple origins gave rise to these intermediates, since three separate regions of the third chromosome chorion locus contained replication bubbles. However, initiation was nonrandom; the majority of initiations appeared to occur near the Bgl II site located between the s18 and s15 chorion genes. The P[S6.9] chorion transposon also contained abundant replication intermediates in follicle cells from a transformed line. Initiation within P[S6.9] occurred near two previously defined cis-regulatory elements, one near the same Bgl II site (in the AER-d region) and one near the ACE3 element.     

Positive and negative DNA elements of the Drosophila grimshawi s18 chorion gene assayed in Drosophila melanogaster.

Germ line transformation has been used to map the cis regulatory DNA elements responsible for the precise and evolutionarily stable developmental expression of the s18 chorion gene. Constructs containing chimeric combinations of Drosophila melanogaster and D. grimshawi DNA regions, as well as D. grimshawi sequences alone, can direct expression in the follicular epithelium, in an s18-specific temporal and spatial pattern. The results indicate that both positive and negative regulatory elements can function when transferred from D. grimshawi to D. melanogaster. The first ca. 100 bp of the 5'-flanking DNA region constitute a minimal, developmentally regulated promoter, expression of which is inhibited by the next 100-bp DNA segment and activated by positive elements located further upstream. Expression of the minimal promoter can also be enhanced by more distant chorion regulatory elements, provided the inhibitory DNA segment is absent.