A Fluorescent Quantitative PCR Approach to Map Gene Deletions in the Drosophila Genome

Corresponding author: Pei-Wen Chiang, Department of Pediatrics, University of Michigan, 1150 W. Medical Center Dr., 3520 MSRB I, Ann Arbor, MI 48109-0650., pwchiang{at}umich.edu (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

We report the application of TaqMan quantitative PCR (QPCR) to map Drosophila chromosome deficiencies by discrimination of twofold copy number differences. For a model system, we used this technology to confirm the X chromosomal mapping of Dspt6 given the autosomal mapping of Dspt4. We then used this technique on both preexisting deletion mutant flies and flies that we generated with deletions to demonstrate the presence or absence of Dspt6, Dspt4, and swa in various deletion mutant flies. In contrast with in situ hybridization studies, QPCR both vitiates the need to do these more intricate studies, and it is more accurate as the site of deletion can be known down to the 10²-bp level. We then successfully applied the technique to the analysis of transcription, demonstrating that the amount of Dspt6 or Dspt4 transcriptional product depended directly on the dosage of the Dspt6 or Dspt4 gene, respectively. The rapidity and precision of this method demonstrates its applicability in Drosophila genetics, the rapid and accurate mapping of Drosophila deletion mutants.

ONE of the major strengths of Drosophila genetics is the availability of flies with chromosomal deficiencies. Such stocks have been collected over the past several decades. Most of these chromosomal deficiencies have been roughly mapped physically and/or genetically. Since these deficiencies were mapped using only a limited number of markers, the region of interest is only approximate. Furthermore, mastery of the cytogenetic and in situ hybridization techniques to accomplish this mapping remains laborious. Therefore, the current mapping strategy cannot accomplish precise molecular mapping. Thus, there are compelling reasons to elaborate a rapid and inexpensive technology to accomplish more precise characterization of the location and extent of chromosomal deficiencies in Drosophila.

We aim to study the biological functions of the chromatin proteins SPT4 and SPT6. Genetic data obtained in yeast suggest that SPT4 and SPT6 are involved in transcriptional regulation and chromatin structure (WINSTON 1992 ). In addition, genetic studies suggest that SPT4 and SPT6 are required for normal recombination (MALAGON and AGUILERA 1996 ), chromosome segregation (BASRAI et al. 1996 ), and transcription elongation (HARTZOG et al. 1998 ). Dspt6 and Dspt4 are the Drosophila homologues of the yeast chromatin proteins SPT6 and SPT4, respectively.

Here, we describe the use of a rapid, facile TaqMan quantitative PCR (QPCR) strategy to map Dspt4 and Dspt6 in deficiency flies. The success of our mapping strategy provides a convenient tool for the mapping of genes of interest in deficiency flies.

	MATERIALS and METHODS

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

TaqMan QPCR:
The TaqMan assay has been described previously (HIGUCHI et al. 1992 ; LIVAK et al. 1995 ; HEID et al. 1996 ). The TaqMan assay requires a forward primer, a reverse primer, and a probe that hybridizes between the forward and reverse primers. The TaqMan PCR technology exploits the 5'-3' nuclease activity of Taq DNA polymerase, allowing direct detection of the PCR product by the uncoupling of a reporter dye from its quencher dye during PCR. The probe consists of an oligonucleotide with a 5' reporter dye, 6-carboxyfluorescein (6-FAM), and a 3'-quencher dye, 6-carboxytetramethylrhodamine (TAMRA). When the probe is intact, the fluorescence from the reporter dye is suppressed by the proximity of the quencher dye. During PCR, if the target is present, the probe specifically anneals between the forward and reverse primer sites. Under this condition, the 5'-3' exonuclease activity of Taq DNA polymerase cleaves the oligonucleotide probe that links the reporter and quencher. The extent of digestion, which depends directly on the amount of PCR that occurs, can be quantified accurately by measuring the increment in fluorescence that results from decreased energy transfer between the reporter and quencher fluors.

The ABI PRISM 7700 can both perform and measure the PCR reaction in real time. This sensitive measurement allows detection of the PCR reaction in the exponential phase that is required for determination of initial genomic sequence copy number. To measure the relative genomic copy numbers of two sequences in a given DNA sample, the quantity of PCR product from the first sequence with the given DNA (whose copy number is to be determined) is divided by the quantity of PCR product from the second sequence (whose copy number is known). This obviates the requirement to know the exact concentration of DNA used for the PCR reaction. The TaqMan QPCR primers and probes were designed using the Primer Express software (Perkin Elmer, Norwalk, CT) and synthesized by Perkin Elmer. The Dspt6 forward primer was 5'-GCCGCACTCCATGAATCTG-3', and the reverse primer was 5'-CATTCACTTCGCCGCTCCTA-3'. The TaqMan probe was 5'-6-FAM-ATGCCACGCCCCTCTACGACGAG-TAMRA3'. The Dspt4 forward primer was 5'-CTCGTGGTATCTATGCCATTTCTG-3', and the reverse primer was 5'-TCCACGATTCTTCATGTCACGTA-3'. The TaqMan probe was 5'-6-FAM-TCCGGAACACTGCCGCAGTCTACC-TAMRA. The swa forward primer was 5'-CGCTGCCGTAATGGTGATC-3', and the reverse primer was 5'-GGCATCGCAGGCAAACC-3'. The TaqMan probe was 5'-6-FAM-TTGAACCGCAGCAATGTCACATACAAGG-TAMRA-3'. The Dspt6 forward primer for reverse transcription (RT)-QPCR was 5'-GGAGAATCTGGGCGTCAAAGT-3', and the reverse primer was 5'-CGCTTTCGTTGTCGTGGAT-3'. The TaqMan probe was 5'-6-FAM-AGACGCTTGAACCGCCGTCTCTCG-TAMRA-3'. The Dspt4 forward primer for RT-QPCR was 5'-TTGACGCGATACCCAAGGAT-3', and the reverse primer was 5'-CTAGTGTGATCATAGACATTGTCCTTGTT-3'. The TaqMan probe was 5'-6-FAM-CTCAAATTGATCAAAACTCTTCACTAGGGAGCAAA-TAMRA-3'. The TaqMan RT-QPCR and Core Reagent PCR kits (with MultiScribe reverse transcriptase and AmpliTaq Gold enzyme) were used for the analysis. The TaqMan RT-QPCR and QPCR reactions were performed on the ABI PRISM 7700 sequence detection system, using conditions suggested by Perkin Elmer. DNA samples for the PCR analysis were isolated by proteinase K/SDS overnight treatment followed by phenol/chloroform extraction. Ten nanograms of DNA was used for the highest concentration of each dilution. RNA samples for the PCR analyses were isolated from adult female flies using the TRIzol method (GIBCO-BRL, Gaithersburg, MD). Total RNA (1 ng) was used for the highest concentration of each dilution in the RT-PCR reaction.

Fly strains:
Df(1)JF5/FM7, Df(1)G4e^LH24i^R/FM7a, Df(1)N73/FM6, and Df(1)5D/FM6 were obtained from the Bloomington Stock Center. y¹ w¹; P{w^+mC = EP}EP2604/CyO was obtained from the Berkeley Drosophila Genome Project stock center. Flies were handled according to standard procedures. Flies with Dspt4 deficiencies were generated by the imprecise excision of a revertible P-element insertion in y¹ w¹; P{w^+mC = EP}EP2604/CyO. y¹ w¹; P{w^+mC = EP}EP2604/CyO was mobilized to generate imprecise excisions as follows: y¹ w¹; P{w^+mC = EP}EP2604/CyO males were crossed to y¹ w¹; Ki¹ P{ry^+t7.2 = Delta2-3}99B/TM3, Sb¹ females. y¹ w¹; P{w^+mC = EP}EP2604/+; Ki¹ P{ry^+t7.2 = Delta2-3}99B/+ males were individually crossed to y¹ w¹; T(2;3)ap^Xa/CyO females. Individual white-eyed y¹ w¹; Df/CyO males were crossed back to y¹ w¹; T(2;3)ap^Xa/CyO females to establish stocks. Stocks lacking non-Curly flies were kept for further analysis.

	RESULTS

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

Testing the TaqMan QPCR mapping strategy:
We had previously developed a fluorescent QPCR-based method to determine gene copy number (CHIANG et al. 1996 , CHIANG et al. 1999 ; CAIRNS et al. 1998 ). Given the need for rapid and accurate mapping of Drosophila deficiency chromosomes, we tested the TaqMan QPCR methodology (HIGUCHI et al. 1992 ; LIVAK et al. 1995 ; HEID et al. 1996 ) for this purpose. We first tested the TaqMan QPCR strategy on the dosage of the sex chromosomes, requiring differentiation between one (male) and two (female) copies per genome. TaqMan QPCR was performed on the samples using two different sets of primers, one set for the X-linked gene, Dspt6, and one set for the autosomal gene, Dspt4. The ratio of QPCR for Dspt6/Dspt4 reflects the copy number ratio of the X-linked gene, Dspt6, to the autosomal gene, Dspt4. This ratio should be 1 for wild-type female flies and 0.5 for wild-type male flies. This is the case, as demonstrated by the control experiments in Table 1A. Each control consisted of a set of four serial dilutions assayed for both Dspt6 and Dspt4. TaqMan QPCR successfully detected a twofold difference between the dilutions. Furthermore, as expected, the Dspt6/Dspt4 ratio for the wild-type female fly was 1, and the ratio for the wild-type male fly was 0.5 (Table 1A). We then tested a known deletion of the swallow (swa) gene on the X chromosome. swa was previously shown to be deleted in Df(1)JF5 (STEPHENSON and MAHOWALD 1987 ). As shown by our TaqMan QPCR analysis, the Dspt4/swa ratio for the Df(1)JF5 female was 2, and the Dspt4/swa ratio for the FM7 male was also 2 (Table 1B). These control experiments showed that TaqMan QPCR is suitable for determining copy number in Drosophila.

TaqMan QPCR mapping of Dspt6-deficient flies:
Dspt6 mapped between 5E1 and 5E8 on the X chromosome (P.-W. CHIANG, unpublished result). According to FlyBase (http://flybase.bio.indiana.edu:82/maps/fbgrmap.html), the 5E6 to 5E8 region is deleted in the deficiency fly Df(1)JF5, 5E3 to 5E8 to 6B is deleted in the deficiency fly Df(1)G4e^LH24i^R, 5C2–5D6 is deleted in the deficiency fly Df(1)N73, and 5D1 to 5E is deleted in the deficiency fly Df(1)5D. We applied TaqMan QPCR to female Df(1)JF5, Df(1)G4e^LH24i^R, Df(1)N73, and Df(1)5D flies. We also tested the nonfertile FM6/FM6 female and the FM6 male. The Dspt6/Dspt4 ratio was close to 1 for female Df(1)JF5, Df(1)G4e^LH24i^R, Df(1)N73, and FM6/FM6 flies, and the Dspt6/Dspt4 ratio was close to 0.5 for male FM6 flies. However, the Dspt6/Dspt4 ratio was close to 0.5 for female Df(1)5D flies, showing a deletion of Dspt6 in Df(1)5D (Table 1C). Thus, TaqMan QPCR mapping placed Dspt6 in the 5E1 to 5E6 region covered by the deletion in Df(1)5D.

TaqMan QPCR mapping of Dspt4-deficient flies:
According to the BDGP database, a P element is inserted into an intron of Dspt4 in EP(2)2604 (P.-W. CHIANG, unpublished result). EP(2)2604 was mapped between 49B7 and 49B11. The homozygous P-element insertion flies are semilethal. Two Dspt4 deficiency flies, Df(2)Dspt4-11 and Df(2)Dspt4-14, were generated through imprecise P-element excision from the EP(2)2604 fly. Both Df(2)Dspt4-11 and Df(2)Dspt4-14 could not complement EP(2)2604. We then applied TaqMan QPCR to female and male Df(2)Dspt4-11 and Df(2)Dspt4-14 flies. In both cases, the Dspt6/Dspt4 ratio for the female fly was close to 2, and this ratio for the male fly was close to 1 (Table 1D). Therefore, TaqMan QPCR confirmed that one copy of Dspt4 is deleted in both heterozygous deficiency flies.

Analysis of transcriptional dosage by TaqMan RT-QPCR:
TaqMan RT-QPCR was performed to determine whether the genomic deletion of Dspt4 or Dspt6 in the deficiency flies resulted in transcriptional dosage. In this experiment, we measured the relative amount of Dspt4 mRNA against the amount of Dspt6 mRNA. To avoid contamination from any genomic DNA present in the RNA preparation, primer sets specific for Dspt4 and Dspt6 mRNA were used in the analysis. These sets used at least one primer bridging two adjacent exons of the gene, eliminating amplification by genomic sequences containing the intervening intron. As expected, no amplification was detected using a DNA template for either primer set (data not shown). As shown in Table 2, the mRNA-derived ratio of Dspt6/Dspt4 was close to 1 for the wild-type fly. The Dspt6/Dspt4 ratio was close to 2 for Df(2)Dspt4-11 and Df(2)Dspt4-14 flies. This result was independent of the sex of the fly that was utilized, reflecting dosage compensation for the Dspt6 gene on the X chromosome. Furthermore, the Dspt6/Dspt4 ratio was close to 0.5 for the Df(1)5D fly. This RT-QPCR analysis confirmed that the deletion of a single genomic copy of Dspt4 or Dspt6 in either of these deficiency flies resulted in a concomitant decrease of Dspt4 or Dspt6 mRNA transcript.

	DISCUSSION

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

Because this is the first application of TaqMan QPCR for Drosophila, we tested several factors essential for the success of this mapping strategy. Dosage for the X-linked gene, Dspt6, with a known copy number difference between males and females, was repeated four times at different dilutions, and the data were highly reproducible (Table 1A). In this analysis, twofold dilutions of each sample were tested, and the technique readily detected a twofold difference. As expected, the relative ratio of Dspt6/Dspt4 for a wild-type female is 1, and the relative ratio of Dspt6/Dspt4 for a wild-type male is 0.5. We also tested a known deletion of the swa gene on the X chromosome (STEPHENSON and MAHOWALD 1987 ), and TaqMan QPCR successfully detected the deletion (Table 1B). Thus, TaqMan QPCR is highly reproducible with the ability to resolve twofold differences in Drosophila.

We then used TaqMan QPCR to determine the copy number of Dspt6 in specific flies. The transmission rate of Dp(1;f)J21A through females to progeny is 28%, and Df(1)JF5 weakly increases this transmission rate (COOK et al. 1997 ). Furthermore, yeast SPT6 was shown to be involved in faithful chromosome segregation (BASRAI et al. 1996 ). This suggested that Dspt6 might be deleted in the deficiency line Df(1)JF5. However, our TaqMan QPCR mapping data demonstrated that Dspt6 is not deleted in Df(1)JF5. We also showed that there is no Dspt6 deletion in Df(1)G4e^LH24i^R, Df(1)N73, and FM6/FM6. In contrast, Dspt6 is deleted in Df(1)5D. This placed Dspt6 between 5E1 and 5E6, illustrating the utility of developing the QPCR method as a fast and accurate way to map quantitative alterations in the Drosophila genome.

To further illustrate the applicability of TaqMan QPCR for mapping, we analyzed the relative ratio of Dspt6/Dspt4 for two deficiency flies that we constructed. Df(2)Dspt4-11 and Df(2)Dspt4-14 were generated through imprecise P-element excision from EP(2)2604. The homozygous deficiency is lethal for both the Df(2)Dspt4-11 and Df(2)Dspt4-14 flies, and both deficiency flies could not complement EP(2)2604. Our TaqMan QPCR mapping demonstrates the deletion of Dspt4 in these two flies, consistent with the complementation data.

The amount of transcript generated from any specific gene generally falls within the limits of a direct correlation with its chromosomal dose. With a direct correlation, a chromosomal reduction would result in 50% of normal expression. From our TaqMan RT-QPCR analysis, the regulation of Dspt4 or Dspt6 expression correlated directly with their gene dosage. A reduction of 50% from the normal expression level of Dspt4 occurred in both Df(2)Dspt4-11 and Df(2)Dspt4-14, and a reduction of 50% from the normal expression level of Dspt6 occurred in Df(1)5D (Table 2).

This TaqMan QPCR-based approach will be useful to establish sequence copy number in the Drosophila genome. Although this task can also be accomplished by in situ hybridization, the QPCR mapping approach is easier, faster, cheaper, and detects deletions at the 10²-nucleotide level rather than at the 10³-nucleotide level required for in situ hybridization. Thus, the molecular order of gene deletions could be determined more precisely in any deficiency fly using the TaqMan QPCR approach. This approach will become even more useful as the genomic sequences are elaborated by the Drosophila Genome Project. The need for a simple and efficient method to map deficiency flies more precisely down to the level of specific genes will become especially useful. Using Dspt4, Dspt6, and swa, we demonstrated that the TaqMan QPCR approach represents a valuable tool for merging this initiative with the mapping of gene deletions in deficiency flies. Furthermore, TaqMan RT-QPCR represents a facile tool for monitoring the expression of genes associated with perturbations resulting in changes of gene copy number.

Manuscript received June 14, 1999; Accepted for publication July 12, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS and METHODS RESULTS DISCUSSION LITERATURE CITED

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