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First published online April 22, 2004
doi: 10.1242/10.1242/dev.01064

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Sequence requirements for function of the Drosophila chorion gene locus ACE3 replicator and ori-ß origin elements

Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-1340, USA

View larger version (42K): [in a new window]   Fig. 2. Sequence requirements for ACE3 function in amplification. (A) The BP construct and ACE3 deletion mutants. For convenience, ACE3 (751 to 1071 in Fig. 1) is numbered from 1 to 320. The shaded regions are conserved among four Drosophila species. The region was identified by sequence analysis and has homology to the ß region in ori-ß. The two Myb consensus binding sites are indicated by stars with one of them outside the conserved shaded regions. The three p120-binding regions are indicated by black bars, the sizes of which are not to scale (Beall et al., 2002). (B) Southern blot analysis of amplification levels for representative independent transgenic lines. BP and ACE3 total deletion (AD) lines were analyzed as controls in the same experiment. rDNA was used as the loading control. (C) Quantitation of three independent assays for each independent transgenic line according to the measurement of amplification levels in Materials and methods. Average and standard deviation are presented in the bar graph. The average fold amplification level is given below the name of the construct. P values are presented for a comparison of each construct to BP using unpaired, two-sided t-tests.

View larger version (34K): [in a new window]   Fig. 4. The 5' 140 bp sequence of ori-ß is required for activity. (A) BP and ori-ß mutants 1-5. For convenience, ori-ß (2258 to 3098 in Fig. 1) is numbered from 1 to 840. The ß-region is indicated by shading. (B) Quantitation of amplification levels for independent transgenic lines.

View larger version (30K): [in a new window]   Fig. 5. The A/T-rich ß region of ori-ß is required for activity. (A) BP and ori-ß mutants 6-9. (B) Quantitation of amplification levels for independent transgenic lines.

View larger version (15K): [in a new window]   Fig. 1. Organization of the third chromosome chorion gene cluster and amplification regulatory elements. The chorion genes are indicated by arrows. Stimulatory regions (`amplification enhancing regions' or AERs) are indicated by hatched boxes. The ACE3 and ori-ß elements, which are necessary and sufficient for amplification, are indicated by black boxes. Evolutionarily conserved sequences within ACE3, regions of homology between ACE3 and ori-ß, and the location of the and ß sequence elements are indicated. All numbering is relative to the published sequence for the 3.8 kb SalI fragment of the third chromosome chorion gene locus (Levine and Spradling, 1985).

View larger version (22K): [in a new window]   Fig. 3. Yeast origin ARS1 and its B2 element cannot functionally replace ori-ß. (A) Yeast origin ARS1 and its DNA unwinding element B2 were cloned into the BP construct replacing the entire ori-ß to generate constructs BP-ARS1 and BP-B2, respectively. (B) Quantitation of amplification levels for independent transgenic lines. BP, Big Parent line 3; OD, the ori-ß total deletion line 1.

View larger version (24K): [in a new window]   Fig. 6. The minimal replicons are not able to support amplification. (A) BP and the minimal replicon constructs. The 2.4 kb fragment containing ACE3, S18 and ori-ß in BP construct was replaced by the indicated combinations of ori-ß mt-2 and/or ACE3 mt-3. (B) Quantitation of amplification levels for independent transgenic lines.

View larger version (148K): [in a new window]   Fig. 7. Anti-ORC2 antibody staining and BrdU labeling in follicle cells. All images were generated using the confocal microscope. Background was intentionally increased in images F, G, I and L to reveal presence or absence of faint staining patterns. (A) Wild-type with anti-ORC2. (B) The transgenic line for BP construct with anti-ORC2. (C) The transgenic line for Yes-3.8S construct with anti-ORC2. (D,E) The transgenic lines for pCaryos-3.8S construct with anti-ORC2, lines 1 and 3, respectively. Line 1 amplifies only to very low level due to genomic position effects, while line 3 amplifies to high level, as shown in Fig. 8. (F) k43fs293/k431 with anti-ORC2. (G) satin with anti-ORC2. (H) chiffon null mutant, genotype chiffonwf24 /Df(2)RA5, with anti-ORC2. (I) chiffon null mutant with anti-ORC2 as in H. (J) Wild type with BrdU labeling. (K) chiffon null mutant, genotype chiffonwf24 /Df(2)RA5, with BrdU labeling. (L) chiffon null mutant, genotype chiffonwf24 /Df(2)RA5, with BrdU labeling as in K. The circles indicate the nuclei.

View larger version (21K): [in a new window]   Fig. 8. Comparison of amplification levels among different transgenic lines. (A) BP, pYes-3.8S and pCaryos-3.8S constructs. (B) Quantitation of amplification level for representative transgenic lines. (C) Amplification levels of endogenous third chromosome chorion gene locus and transgenic constructs in wild-type and k43 mutant backgrounds. All transgenic constructs are homozygous. Y8, Yes-3.8S construct transgenic line 8; k43/TM6B, heterozygous (non-mutant) background; k43/k43, mutant background k43fs293/k431; E, endogenous amplification level; T, transgene amplification level. Average fold amplification levels are presented below the graph.

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