The genetic interval 35C to 36A on chromosome arm 2L of Drosophila melanogaster has been saturated for mutations with visible or lethal phenotypes. 38 loci have been characterized, including several maternal-effect lethals (vasa, Bic-C, chiffon, cactus and cornichon) and several early embryonic lethals, including snail and fizzy. About 130 deletions have been used to order these loci. Complex interactions between mutant alleles have been uncovered in the immediate genetic environs of the snail gene, as has further evidence for an interaction between this region and that including the nearby genes no-ocelli and elbow (Ashburner, 1990).

chiffon mutants were generated by Schüpbach and co-workers as part of a screen for female sterile mutations on the Drosophila second chromosome (Schüpbach, 1991). The homozygous chiffon phenotype is female sterile, characterized by a thin, fragile chorion (eggshell) structure that suggested the name for the gene. The similarity of the chiffon thin chorion phenotype to the thin chorion of k43 and other genes disrupting chorion gene amplification suggests that chiffon might also disrupt amplification. The phenotypes of multiple chiffon alleles were characterized. chiffon phenotypes fall into two general classes: a mild phenotype where defects are limited to thin chorions, and a severe phenotype characterized by thin chorions, as well as rough eyes and thin thoracic bristles. All homozygous alleles exhibit the mild phenotype with the exception of chiffon^WF24 homozygotes, which exhibit the severe phenotype. When placed over deficiency, the chiffon^WD18 mild allele then exhibits the severe phenotype, characterized by rough eyes and thin thoracic bristles. Thus the mild phenotype appears to be hypomorphic. The phenotype of chiffon^WF24 does not change over deficiency, suggesting that the chiffon^WF24 severe phenotype represents the null. This has been confirmed by molecular characterization of chiffon^WF24 and another null allele, chiffon^ETBE3 (Landis, 1999).

The effect of chiffon mutations on chorion gene amplification has been determined by Southern analysis of DNA isolated from mutant egg chambers. DNA was isolated from mutant and control stage 13 egg chambers, and the samples were divided equally onto identical slot blots. One blot was hybridized with a probe specific for the amplified third chromosome chorion gene cluster, and the other with a probe specific for the non-amplified rDNA genes. DNA isolated from male flies, where amplification does not occur, yields approximately equal signal with both probes. In contrast, DNA isolated from non-mutant control egg chambers exhibits greatly increased signal with the chorion probe, due to amplification. In DNA isolated from chiffon mutant egg chambers, hybridization to the chorion probe is reduced to nearly non-amplified levels (Landis, 1999).

The developmentally regulated amplification of the Drosophila third chromosome chorion gene locus requires multiple chromosomal elements. Amplification control element third chromosome (ACE3) appears to function as a replicator, in that it is required in cis for the activity of nearby DNA replication origin(s). Ori-ß is the major origin in the locus, and is a sequence-specific element that is sufficient for high-level amplification in combination with ACE3. Sequence requirements for amplification were examined using a transgenic construct that was buffered from chromosomal position effects by flanking insulator elements. The parent construct supported 18- to 20-fold amplification, and contained the 320 bp ACE3, the ~1.2 kb S18 chorion gene and the 840 bp ori-ß. Deletion mapping of ACE3 revealed that an evolutionarily conserved 142 bp core sequence functions in amplification in this context. Several deletions had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. S. cerevisiae ARS1 origin sequences could not substitute for ori-ß, thereby confirming the sequence specificity of ori-ß. Deletion mapping of ori-ß identified two required components: a 140 bp 5' element and a 226 bp A/T-rich 3' element called the ß-region that has significant homology to ACE3. Antibody to the origin recognition complex subunit 2 (ORC2) recognizes large foci that localize to the endogenous chorion gene loci and to active transgenic constructs at the beginning of amplification. Mutations in Orc2 itself, or the amplification trans regulator satin eliminated the ORC2 foci. By contrast, with a null mutation of chiffon (dbf4-like) that eliminates amplification, diffuse ORC2 staining was still present, but failed to localize into foci. The data suggest a novel function for the Dbf4-like Chiffon protein in ORC localization. Chromosomal position effects that eliminated amplification of transgenic constructs also eliminate foci formation. However, use of the buffered vector allowed amplification of transgenic constructs to occur in the absence of detectable foci formation. Taken together, the data suggest a model in which ACE3 and ori-ß nucleate the formation of an ORC2-containing chromatin structure that spreads along the chromosome in a mechanism dependent upon Chiffon (Zhang, 2004).

The use of insulator elements, the suppressor of Hairy-wing protein binding sites [su(Hw)BSs], protects transgenic chorion gene constructs from chromosomal position effects and allows for detailed analysis of sequence requirements for amplification. The ACE3 replicator and ori-ß origin elements are necessary for efficient amplification. A construct containing only the 320 bp ACE3 and the 840 bp ori-ß ('Small Parent' or SP) demonstrates that these elements are also sufficient for amplification; however, the levels of amplification are moderate and are subject to significant chromosomal position effects even in the presence of the flanking insulator elements. In the BP construct, the 320 bp ACE3 and the 840 bp ori-ß are in their normal context, i.e., spaced by the ~1.2 kb S18 chorion gene, and these sequences supported efficient amplification (~20 fold) with minimal position effects. For this reason, the BP construct was chosen for detailed analysis of ACE3 and ori-ß sequence requirements. Evolutionarily conserved core sequences were found to be sufficient for the majority of ACE3 activity. Deletion of the less conserved 5' and 3' flanking sequences within ACE3 had quantitative effects, suggesting that multiple, partially redundant elements comprise ACE3. These results and conclusions are analogous to those from a previous study of ACE3 sequence requirements done in the context of a larger, unbuffered construct. No deletion of a subset of ACE3 sequences reduced amplification to the extent of a deletion of all of ACE3. The sequence requirements for ACE3 function in amplification defined in this study correlate well with the sequence requirements previously defined for ORC binding in vitro. The central region of ACE3, corresponding to the evolutionarily conserved sequences, is most crucial for ORC binding, while the 5' and 3' flanking regions within ACE3 stimulates ORC binding. Taken together, the data suggest that the multiple, partially redundant elements that comprise ACE3 are ORC binding sites, and that one crucial function of ACE3 in amplification is to bind ORC (Zhang, 2004).

Recently a protein complex containing Drosophila Myb, p120 and three other proteins was found to bind to both ACE3 and ori-ß sequences, and Myb was found to be required in trans for amplification. Both Myb and p120 are capable of DNA binding on their own, and have binding sites that overlap with the essential core region of ACE3. There are two Myb consensus binding sites (positions 121 to 127 and 137 to 142) and three p120 binding regions (27 to 56, 89 to 105 and 184 to 216) in ACE3 element. Small (30-40 bp) deletions that removed one of Myb consensus binding sites or one of the p120-binding sites in the core region of ACE3 had negative effects on amplification in the context of the BP construct. Taken together, these data suggest that another function of the conserved core region sequences of ACE3 is to bind the Myb complex (Zhang, 2004).

Two-dimensional gel analyses of the endogenous third chromosome chorion gene locus demonstrate that the majority (70%-80%) of initiations occur in a region containing the ori-ß element. In 2D gel analysis of the BP construct, abundant initiation events, as indicated by bubble structures, were associated with the ori-ß element while no initiations could be detected for ACE3. To begin to examine the sequence requirements for ori-ß function, ori-ß was substituted by either the entire 193 bp S. cerevisiae ARS1 origin sequence, or the 20 bp B2 element from ARS1, which is a putative DNA unwinding element. No activity in supporting amplification was detected for either fragment, indicating that ori-ß is not simply an A/T-rich or easily unwound sequence. Deletion mapping suggests two sub-components of ori-ß: an essential 5' 140 bp region that is not particularly A/T-rich, and the 226 bp A/T-rich ß region. The 366 bp fragment containing both regions is sufficient for the majority of ori-ß activity. In addition the 3' most 140 bp of the starting 840 bp ori-ß fragment may have a small stimulatory effect. The portion of the alpha region in ACE3 and the ß region in ori-ß are each A/T-rich and internally repetitive, and have some sequence homology with each other. A large fragment containing the ß-region can bind ORC in vitro. Therefore, it is hypothesized that, like the sequences in ACE3, one required function of the ß region in ori-ß is to bind ORC (Zhang, 2004).

A similar organization has been identified for a developmentally regulated origin in another dipteran fly, Sciara coprophila. In Sciara larvae, the salivary gland cells amplify several loci containing putative pupal case genes, resulting in chromosomal DNA 'puffs'. The ori II/9A DNA replication initiation site has been mapped to the nucleotide level and has similarities to the yeast ARS. Drosophila ORC has been shown (Bielinsky, 2001) to bind to an 80 bp region adjacent to this replication start site (Zhang, 2004).

Analysis of trans-acting gene mutations confirms the intimate association between amplification initiation and the formation of a large focus of ORC2 localization at amplifying chromosomal loci. Mutations in k43 (Orc2) itself, or the newly identified trans-regulatory gene satin, eliminated ORC2 antibody staining and focus formation. Null mutations of chiffon, a dbf4-like gene, completely eliminate amplification. In chiffon-null mutant follicle cells, diffuse ORC2 staining was still present in the nucleus, but it failed to localize into foci at stage 10A. A similar phenotype had previously been observed for mutations in the amplification trans-regulators dDP (a subunit of E2F) and Rbf. A role for chiffon in ORC localization was surprising given the well-characterized order of events known for other organisms. In S. cerevisiae and Xenopus in vitro systems, ORC is bound at origins and is required for the subsequent binding of Dbf4 and its catalytic subunit CDC7, which is one of the last events before origin firing. The data suggest two possible models for the role of chiffon in ORC2 focus formation during amplification. In the first model, Chiffon protein would bind first to the chorion gene sequences, either directly or more likely via an interaction with another DNA-binding protein, since the Chiffon sequence suggests no obvious DNA-binding motifs. Chiffon would then recruit Drosophila ORC2 to the DNA. This model seems unlikely given the opposite order of events observed in yeast and in Xenopus in vitro systems. In the second and favored model, a relatively small amount of ORC binds first to the chorion gene loci, most probably to the conserved core sequences in ACE3 and the ß region in ori-ß. Chiffon protein would then interact with the ORC complex(es) and catalyze the further binding of large amounts of ORC to generate the dramatic foci observed upon staining with ORC2 antibody. A mechanism is envisioned in which the alpha and ß regions nucleate ORC binding, and then through a process dependent upon Chiffon, an ORC-containing chromatin structure spreads along the chromosome to form the dramatic foci. This model is appealing in that it provides a way for ACE3 and ori-ß to interact and form a chromosomal domain activated for DNA initiation events. Previous data have indicated that ACE3 and ori-ß interact during amplification in a way that can be blocked by an intervening insulator element. Moreover, analysis of the endogenous locus indicates that ACE3 is required for the activation of multiple origins spread throughout a chromosomal domain containing the chorion gene cluster. This model is testable in that it predicts that the insulators would form a boundary for this ORC-containing chromatin structure (Zhang, 2004).

The possibility cannot be ruled out that chiffon is not the true Dbf4 homolog in Drosophila, but this appears unlikely. Chiffon shows conservation with Dbf4 homologs from all other species in the key ORC-binding domain (called CDDN2) and the CDC7-binding domain (called CDDN1). Moreover, there is no other gene in the Drosophila genome with detectable homology to Dbf4. However, Chiffon contains an additional large C-terminal protein domain present only in Dbf4 homologs from closely related species, such as Medfly and mosquito. It is speculated that this C-terminal domain may play a specific role in chorion gene amplification. Further experiments will be required to determine if a role in ORC localization is a characteristic of all Dbf4 family members, or whether this represents a function unique to the large chiffon protein (Zhang, 2004).

Consistent with the correlation between ORC2 focus formation and amplification initiation, dramatic ORC2 foci can form at the sites of amplifying transgenic chorion gene constructs. It was therefore surprising that in no cases were foci observed at the sites of actively amplifying BP constructs. This is despite the fact that amplification was readily observed at these sites by BrdU incorporation. One possible explanation might be the moderate amplification level of BP (18- to 20-fold). However, the YES-3.8S construct amplifies to similar levels as BP, and an extra ORC2 focus was observed for every line. In addition multimers of ACE3 with very low amplification levels are capable of creating additional ORC2 foci. Therefore, the lack of focus formation with BP is not simply due to its moderate amplification level, but must reflect the specific sequence content or arrangement in BP. The lack of focus formation in BP is also not simply due to the presence of flanking insulator elements; the YES-3.8S construct contains the same flanking insulator elements. The data suggest two non-exclusive possibilities. The first is that the difference is due to the fact that BP contains less extensive chorion gene sequences than YES-3.8S. Although deletion of these sequences has no significant effect on amplification level, it may be that redundant ORC binding sites have been deleted, thereby dramatically reducing visible focus formation. The second possibility is that the relevant difference is the amount of sequence present inside the insulators. BP contains only 2.4 kb between the insulators, whereas Yes-3.8 contains 9 kb. If the insulators limit the size of the domain in which an ORC containing chromatin structure can spread from ACE3 and/or ori-ß, then the small size of this domain in BP may not create a visible focus. In this model, the insulators would have two significant effects on amplification: they would prevent the spread of negative chromatin structures into the bounded region and thereby prevent negative chromosomal position effects; and they would limit the ORC containing chromatin structure and initiation activity to the bounded region. These models should be testable in the future by CHIP analysis of chromatin structures associated with chorion gene sequences and transgenic constructs. It will be of interest in the future to determine if su(Hw)BS insulators or other types of insulators are involved in organizing the endogenous chorion gene locus and the rest of the genome into domains of DNA replication activity (Zhang, 2004).

Ashburner, M., et al. (1990). The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. VII. Characterization of the region around the snail and cactus loci. Genetics 126(3): 679-94