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.     

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.     

DNA sequence of a 3.8 kilobase pair region controlling Drosophila chorion gene amplification

During Drosophila oogenesis, two clusters of chorion genes and their flanking DNA sequences undergo amplification in the ovarian follicle cells. Amplification results from repeated rounds of initiation and bidirectional replication within the chorion gene regions, possibly from a single origin, producing nested replication forks. Previously we have shown that following reintroduction into the Drosophila genome, a specific 3.8 kilobase pair DNA segment from the amplified third chromosome domain could induce developmentally regulated amplification at its site of insertion. Here we present the complete nucleotide sequence of this amplification control element and of genes encoding the chorion structural proteins s18-1 and s15-1, which are contained within it. Sequences that may be involved in the regulation of chorion gene amplification and expression are identified.     

Genetic analysis of the gene cluster responsible for synthesis of serotype e-specific polysaccharide antigen in Actinobacillus actinomycetemcomitans

A gene cluster associated with the biosynthesis of the serotype e-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans IDH1705 belonging to serotype e was cloned and sequenced. This cluster consisted of 18 open reading frames. Escherichia coli produced the polysaccharide that reacts with the serotype e-specific antiserum when transformed with a plasmid containing the cluster. Comparing the structure of the gene cluster with similar clusters from A. actinomycetemcomitans strains Y4 (serotype b) and NCTC9710 (serotype c) revealed that a 5.3-kb region containing the distal half of one gene and two entire genes in the cluster from strain IDH1705 replaced a 6.2-kb region containing eight genes in the cluster from strain Y4, and a 4.7-kb region containing four genes in the cluster from strain NCTC9710. These results suggest that this region is essential to the antigenic specificity of serotype e A. actinomycetemcomitans.     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.