Boris C. Dunkov

Cytochrome p450 gene clusters in Drosophila melanogaster

Twelve cytochrome P450 cDNA fragments were cloned fromDrosophila melanogaster by reverse transcriptase/PCR (RT/PCR) using degenerate oligonucleotide primers. The corresponding genes belong to several subfamilies of the CYP4 and CYP9 P450 families. Only two of these genes,Cyp4d1 andCyp4d2, have previously been described.In situ hybridization of each of the cDNA fragments showed two clusters of genes; one near the tip of theX chromosome and the other on the left arm of chromosome2. Interestingly the latter cluster comprises widely divergent genes belonging both to the CYP9 and CYP4 families and also to the CYP6 family (Cyp6a2). Putative allelic variants of several of the genes were found in different insecticide-resistant and -susceptible strains (Hikone R, Haag 79 and Oregon R). The identification of these genes and alleles will allow us to clarify the involvement of P450s in xenobiotic metabolism and will facilitate a genetic analysis of P450 functions in insects.

Drosophila melanogaster ferritin: cDNA encoding a light chain homologue, temporal and tissue specific expression of both subunit types.

Drosophila melanogaster secreted ferritin like the cytosolic ferritins of other organisms is composed of two subunits, a heavy chain homologue (HCH) and a light chain homologue (LCH). We report the cloning of a cDNA encoding the ferritin LCH of this insect. As predicted from the gene sequence, it contains no iron responsive element (IRE). Northern blot analysis reveals two mRNAs that differ in length due to the choice of polyadenylation signals. Message levels vary through the life cycle of the fly and are markedly increased by high levels of dietary iron. The gut is the main site of increased message synthesis and iron preferentially increases the amount of shorter messages. Western blotting reveals that LCH is the predominant ferritin subunit in all life stages. The amount of LCH protein corresponds well with the message levels in control animals, while in iron-fed animals LCH does not increase proportionally with the message levels. In contrast, the amount of HCH is less than that would be predicted from message levels in control animals, but corresponds well in iron-fed animals. Ferritin is abundant in gut and hemolymph of larvae and adults and in ovaries of adult flies. At pupariation, ferritin becomes more abundant in hemolymph than in other tissues.

Aedes aegypti transferrin. Gene structure, expression pattern, and regulation

Mosquitoes and all other insects so far examined have an abundant haemolymph transferrin (Tsf). The exact function of these proteins has not been determined, but they may be involved in iron transport, in oogenesis and in innate immune defence against parasites and pathogens. The Tsf gene of Aedes aegypti has been cloned and sequenced. It contains a single small intron, which contrasts it to vertebrate Tsf genes that contain up to sixteen introns. The promoter region of the gene is rich in putative NF‐κB binding sites, which is consistent with the postulated role of Tsf in insect innate immunity. Tsf message levels are very low in embryos and early larvae, but high in late larvae, pupae and adults. Western blotting experiments revealed high levels of Tsf protein in pupae and adults. Late larvae and ovaries of blood‐fed mosquitoes have little intact protein, but two prominent proteolytic degradation products. These may represent biologically active peptides, as has been shown for other organisms. Tsf message is down‐regulated by inorganic iron in the diet or environment, but up‐regulated by a blood meal in the adult female. The up‐regulation following a blood meal may, in part, be due to the decrease in juvenile hormone (JH) that is known to follow blood feeding. Treatment of blood‐fed females with methoprene, an analogue of JH, resulted in decrease of the Tsf message.

Organization of the ferritin genes in Drosophila melanogaster.

The organization of two closely clustered genes, Fer1HCH and Fer2LCH, encoding the heavy-chain homolog (HCH) and the light-chain homolog (LCH) subunits of Drosophila melanogaster ferritin are reported here. The 5019-bp sequence of the cluster was assembled from genomic fragments obtained by polymerase chain reaction (PCR) amplification of genomic DNA and from sequences obtained from the Berkeley Drosophila Genome Project (BDGP) (http://www.fruitfly.org). These genes, located at position 99F1, have different exon-intron structures (Fer1HCH has three introns and Fer2LCH has two introns) and are divergently transcribed. Computer analysis of the possibly shared promoter regions revealed the presence of putative metal regulatory elements (MREs), a finding consistent with the upregulation of these genes by iron, and putative NF-kappaB-like binding sites. The structure of two other invertebrate ferritin genes, from the nematode Caenorhabditis elegans (located on chromosomes I and V), was also analyzed. Both nematode genes have two introns, lack iron-responsive elements (IREs), and encode ferritin subunits similar to vertebrate H chains. These findings, along with comparisons of ferritin genes from invertebrates, vertebrates, and plants, suggest that the specialization of ferritin H and L type chains, the complex exon-intron organization of plant and vertebrate genes, and the use of the IRE/iron regulatory protein (IRP) mechanism for regulation of ferritin synthesis are recent evolutionary acquisitions.