Antony J. Howells

Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration.

The white, brown and scarlet genes of Drosophila melanogaster encode proteins which transport guanine or tryptophan (precursors of the red and brown eye colour pigments) and belong to the ABC transporter superfamily. Current models envisage that the white and brown gene products interact to form a guanine specific transporter, while white and scarlet gene products interact to form a tryptophan transporter. In this study, we report the nucleotide sequence of the coding regions of five white alleles isolated from flies with partially pigmented eyes. In all cases, single amino acid changes were identified, highlighting residues with roles in structure and/or function of the transporters. Mutations in w(cf) (G589E) and w(sat) (F590G) occur at the extracellular end of predicted transmembrane helix 5 and correlate with a major decrease in red pigments in the eyes, while brown pigments are near wild-type levels. Therefore, those residues have a more significant role in the guanine transporter than the tryptophan transporter. Mutations identified in w(crr) (H298N) and w(101) (G243S) affect amino acids which are highly conserved among the ABC transporter superfamily within the nucleotide binding domain. Both cause substantial and similar decreases of red and brown pigments indicating that both tryptophan and guanine transport are impaired. The mutation identified in w(Et87) alters an amino acid within an intracellular loop between transmembrane helices 2 and 3 of the predicted structure. Red and brown pigments are reduced to very low levels by this mutation indicating this loop region is important for the function of both guanine and tryptophan transporters.

Sub-cellular localisation of the white/scarlet ABC transporter to pigment granule membranes within the compound eye of Drosophila melanogaster.

The white, scarlet, and browngenes of Drosophila melanogasterencode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed.

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.

The hermit transposable element of the Australian sheep blowfly, Lucilia cuprina, belongs to the hAT family of transposable elements

We report the cloning ofhermit, a member of thehAT family of transposable elements from the genome of the Australian sheep blowfly,Lucilia cuprina. Hermit is 2716 bp long and is 49% homologous to the autonomoushobo element,HFL1, at the nucleic acid level.Hermit has 15 bp terminal inverted repeats that share 10 bp with the terminal inverted repeats ofHFL1. Conceptual translation reveals a 583 residue open reading frame (ORF) that is 64% similar and 42% identical to theHFL1 ORF. However, the sequence of thehermit element contains two frameshifts within the putative ORF, indication thathermit is an inactive element. Analysis ofL. cuprina strains from within and outside Australia suggested thathermit is present as a single copy in all the genomes analysed.

MOLECULAR CHARACTERIZATION OF THE CINNABAR REGION OF DROSOPHILA MELANOGASTER : IDENTIFICATION OF THE CINNABAR TRANSCRIPTION UNIT

Early studies of eye pigmentation in Drosophila melanogaster provided compelling evidence that the cinnabar (cn) gene encodes the enzyme kynurenine 3-monooxygenase. (EC 1.14.13.9). Here we report the cloning of approximately 60 kb of DNA encompassing the cn gene by chromosome walking in the 43E6-F1 region of chromosome 2. An indication of the position of cn within the cloned region was obtained by molecular analysis of mutants: 9 spontaneous cn mutants were found to have either DNA insertions or deletions within a 5 kb region. In addition, a 7.8 kb restriction fragment encompassing the region altered in the mutants was observed to induce transient cn function when microinjected into cn- embryos. The cn transcription unit was identified by Northern blotting and sequence analysis of cDNA and genomic clones from this region. The predicted cn protein contains several sequence motifs common to aromatic monooxygenases and is consistent with the assignment of cn as encoding the structural gene for kynurenine 3-monooxygenase.

Cloning and characterization of the white and topaz eye color genes from the sheep blowfly Lucilia cuprina.

SummaryClones carrying thewhite andtopaz eye color genes have been isolated from genomic DNA libraries of the blowflyLucilia cuprina using cloned DNA from the homologouswhite andscarlet genes. respectively, ofDrosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of thewhite gene ofL. cuprina has been determined, and the homology to the corresponding region ofD. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing theD. melanogaster genes is free of repeated sequences. that encompassing theirL. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome ofL. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 104 copies to less than 102 copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both thewhite andtopaz gene regions are found within and between populations ofL. cuprina.

Molecular and genetic studies on the euchromatin-heterochromatin transition region of the X chromosome of Drosophila melanogaster

A recombinant Charon 4 bacteriophage has been isolated on the basis of RNAs which are enriched in the head of the adult Drosophila melanogaster and hence are likely to be of neural origin. The cloned insert maps to the near vicinity of the uncoordinated locus in polytene chromosome band 19E8. This band is within the transition zone between the euchromatic and heterochromatic regions of the X chromosome, a region which has been well characterized cytogenetically. The insert contains both repetitious and low copy number sequences, some of which vary extensively in both frequency and restriction fragment size between different laboratory strains. One particular family of moderately repeated sequences occurs predominantly in divisions 19 and 20 of the X chromosome and perhaps the distally located X heterochromatin. The molecular landscape surrounding the initial entry point contains many repeated sequences and is thus unlike those observed in most published chromosomal walks. The possible significance of the presence of repeated sequence families in the distinct properties of this region are discussed.

Characterization and chromosomal distribution of a tandemly repeated DNA sequence from the Australian sheep blowfly, Lucilia cuprina

In the course of making a Lucilia cuprina genomic DNA library, a ladder of bands was seen in partial Sau3A digests. Complete digestion reduced this ladder to predominantly monomer units of approximately 190 bp. Nine independently isolated copies of this repeat were cloned and sequenced. Only two of these isolates are identical in sequence, the most divergent being 71% homologous. This satellite DNA occurs in all three wildtype strains tested, and, for the single case examined, in the embryonic, larval, pupal, and adult DNA. It represents approximately 3%–4% of the genome. Data obtained from in situ chromosome hybridizations indicate that this sequence is concentrated around the centromeric regions of the autosomes and over most of the sex chromosomes. Labelling is much stronger in mitotic compared with polytene chromosomes showing directly that this centromeric satellite DNA is grossly under-replicated during polytenization. This under-replication is even more pronounced on the sex chromosomes compared with the autosomes.

Drosophila melanogaster contains both X-linked and autosomal homologues of the gene encoding calcineurin B

A transcription unit was identified in the 43E polytene band region of the second chromosome of Drosophila melanogaster (Dm) whose putative translation product has 85% amino acid (aa) identity with the B subunit of the calcineurin protein (CnB) from humans. Unlike the previously described intronless Dm CnB gene homologue, which is located within the 4F band region of the X chromosome, the coding region of this second CnB is found to be interrupted by three introns. Conceptual translation of both Dm CnB genes predict proteins of identical size that are 98% identical in aa sequence. Northern blot analyses indicate that Dm pupae and adults express two different CnB-encoding transcripts that are differentially regulated.

Gene and Genome Structure in Diptera: Comparative Molecular Analysis of an Eye Colour Gene in Three Species

Drosophila melanogaster and Drosophila buzzatii belong to the same family (Drosophilidae) within Order Diptera but to different species groups, which are thought to have diverged about 50–60 MY ago; the sheep blowfly (Lucilia cuprina), however, belongs to a different family (Calliphoridae), which is thought to have diverged from Drosophilidae at least 100 MY ago (Beverley and Wilson, 1984). We have been analyzing the similarities and differences in gene and genome organization at the molecular level between these species using eye colour genes as our model system.