Male specific expression of a cytochrome P450 (Cyp312a1) in Drosophila melanogaster

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Abstract

Using cDNA array techniques, the transcriptional levels of Drosophila cytochrome P450 (P450) genes were compared between male and female flies. Of the 86 P450s, 15 isoforms were picked up and the levels of transcription were confirmed by the real time quantitative RT-PCR. Cyp315a1 and Cyp302a1, which had been reported as P450s involved in the ecdysteroid biosynthesis, were included in the 15 isoforms and expression of these P450s was 8.6- and 7.9-fold higher in females than in males, respectively. In addition, we confirmed that expression of Cyp312a1 was 82-fold higher in adult males than females. This gene expression was observed mostly in the abdomen and its transcription level gradually increased from pupal stage and peaked in the 5-day-old adult. Furthermore, the male specific expression of Cyp312a1 was universally observed in three Drosophila strains originated from the USA (Oregon R), China (Canton S), and Japan (HKJ), suggesting possible involvement of this P450 in significant endogenous catalytic reaction(s). This is the first report of a P450 enzyme being predominantly expressed in male Drosophila.

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Materials and methods

Drosophila strains. Three parental strains of D. melanogaster, Oregon R, Canton S, and HKJ were used. HKJ is a field strain collected from Higashi Kurume-shi, Tokyo, Japan, in 2002 using a bait trap with grape peel. All strains were maintained at 25 °C on a standard medium based on the corn meal, glucose, and ebios.

Preparation of P450 cDNAs. cDNAs used for spotting on the cDNA array filter were prepared as follows. Sequences of amino acid and genomic DNA of all cytochrome P450s in D. melanogaster

cDNA array analysis

In total, we spotted cDNAs of 86 P450s and four internal controls. Each luminescence level was calculated based on the expression of the internal β-actin gene since the expression of this gene was least affected by synthesis of large amount of vitellogenin proteins in the ovary of female. Expression patterns are shown in Fig. 1. Cyp4g1 had the highest signal intensities, which was consistent with a previous study [21]. Of the 86 P450s, 17 isoforms were excluded from further analysis due to low

Discussions

Cyp312a1 was found to be expressed about 80-fold higher in adult male than female of Drosophila. This unique phenomenon is conserved among three strains of different origins, suggesting that this P450 is playing an important role, which is characteristic of male. In mammals, liver P450s involved with steroid hydroxylation have been well studied. Several P450s that metabolize sex hormones such as testosterone and estrogen are expressed in a sex-dependent manner [22]. On the contrary, insects do

Acknowledgements

We express our gratitude to Dr. Amanda Persad for the critical correction of the manuscript and to Dr. Noriaki Agui for his helpful comments on the manuscript. We also thank Kazue Ito for her technical support on the rearing of Drosophila melanogaster and to Kyoko Kasai for her help to collect HKJ strain. This work was supported by Grant-in-Aid for The Ministry of Education, Culture, Sports, Science and Technology (14560046).

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