The ebony⁴ mutant of Drosophila melanogaster.

Pérez, M.M., and L.A. Quesada-Allué.  Instituto de Investigaciones Bioquímicas Patricias Argentinas 435- (1405) Buenos Aires, Argentina.  e-mail: lualque@iib.uba.ar

Introduction

Because of its melanic phenotype being easily recognizable in genetic experiments, D. melanogaster ebony is used in many laboratories, mostly in secondary schools and undergraduate teaching at the universities, but also in fundamental. research.  Although well studied, mainly in relation to catecholamine metabolism, there is relatively little recent research on this mutant.  The locus ebony encodes the ß-alanine-dopamine synthase as postulated and indirectly shown by several. authors (Jacobs and Brubaker, 1963;  Hodgetts and Konopka, 1973;  Bruce Black, personal. communication) and demonstrated in our laboratory by measuring directly in vitro the enzymatic activity and the lack of it in the mutant ebony (Pérez et al., 1997).  In spite of the importance of ebony as a key mutant in sclerotization, up to recently, when the locus was cloned and sequenced (Hovemann et al., 1998), nothing was known on the characteristics of the ebony gene.

Methods

RNA was extracted with TRIzol (GIBCO), purified and used to synthesize cDNA with reverse transcriptase and specific primers.  Amplification was with a combination of specific primers named N-TERM, SGST, EHQR, KIRGH (according to the putative amino acid sequence of specific ebony regions) and C-TERM.  After electrophoresis, the DNA bands were eluted with Gene Clean (Bio 101), cloned in vector T (Promega) and sequenced.  Sequence analysis was made with the programs Lasergene (DNA Star) or Blast.  The amplified sequences were also cloned in the expression vector pET22b(+) (Novagen).  Radioactive probes (> 1´ 10⁹ cpm/mg) were generated and labeled by random priming with the Prime-a-Gene Labeling System of Promega containing as DNA polymerase a DNA I Klenow fragment.  After purification the probes were hybridized (62^oC o/n) with the Hybond-N (Amersham) membranes containing the mRNAs transferred from agarose electrophoresed gels (see legend to Figure 2).  After several. washes the Northern blots stained with Methylene Blue were subjected to autoradiography (72 h, -70^oC).

Results and Discussion

As partially shown in Figure 1A, starting with cDNA and appropriate primers, we have cloned and sequenced again the ebony gene of wild type Oregon R, as well as for the first time, the ebony⁴ mutant.  The wild type sequence was fully (more than 99%) coincident with the original.

 

(A)  Scheme of wt and e4 exons/introns. Exons:  I = 1108 bp,  II = 168 bp, III = 772 bp,  IV = 91 bp,  V = 176 bp,  VI =322 bp. (Total: 2637 bp ó 879 aa). Introns:  a = 65 bp, b =  86 bp,  c = 66 bp, d = 60 bp,  e = 55 bp (Total: 336 bp).	(B)  Position of e4 deletion within exon I.
Figure 1:  Coding region of ebony  and  ebony4  deduced from cDNA sequence.

report by Hovemann et al. (1998) and with the recent publication of the full  D. melanogaster genome (Adams et al., 2000).  The gene sequence showed by Hovemann et al. (7815 bp starting 4634 bp before AUG) shows six exons (2631 bp) and five introns (336 bp)  (see sizes in Figure 1) (Hovemann et al., 1998).  According with our results, ebony⁴ exhibited a gap of 448 bp (between bases 623 and 1071 of the wt cDNA sequence, see Figure 1B), thus generating a shorter transcript.  From the published D. melanogaster genome, it is assumed that only one copy of ebony is present in the genome, in agreement with the well-known fact that no similar biochemical. phenotype has been found in any other melanic mutant.  The theoretical. size of the mutant transcript as judged from Northern blots (around 2.7 Kb, Figure 2A) agrees well with the genomic sequence (around 3.2 Kb).  No alternatively spliced mRNA species were detected in the wild type or in the mutant using flies from larval. to adult stages.  As previouslyreported, the mutant e⁴ protein extract showsless than 0.5% of the activity of the wild type (not shown), according to our standard assay for in vitro synthesis of N-ß-alanyldopamine (Wappner et al., 1996a;  Pérez and Quesada-Allué, 1998).  Therefore, we might assume that either the missing portion of exon contains the active site of the enzyme or that the conformational change abolishes the catalytic ability, or both.  As found in the GenBank, there is a faint similarity, in terms of sequence and function, with a bacterial peptide synthase, but nothing can be inferred with respect to the active site.  Surprisingly, so far we have been unable to identify a sequence similar to that of ebony in Ceratitis capitata, where the ß-alanine-dopamine synthase is assumed to be encoded by the gene niger (Wappner et al., 1996a;  Wappner et al., 1996b).

Figure 2:  Northern blot. Total RNA from wt and e4 were isolated using TRIzol(r) Reagent (Gibco BRL). For Northern blot analysis, equal amount of total RNA (20 mg) were electrophoresed on 1% agarose gel containing 2.2 M formaldehyde as described in Sambrook et al., 1989). adult: RNA isolated from just eclosed adults, pharate adults: RNA isolated from late pharate adults, thorax & abdomen: RNA isolated from thorax and abdomen of old adults  heads: RNA isolated from the corresponding heads of the same old adults (1-2 weeks old). M: RNA markers A)   Autoradiography with the radioactive probe. Two probes were used: one from base 1 (initial codon, first exon) up to base 1377 (third exon) and the other from base 1357 (third exon) up to base 2631 (last codon sixth exon)  B)   Methylene blue staining of 18s rRNA used to quantify sample loading.

References:  Jacobs, M.E., and K.K. Brubaker 1963, Science 139: 1282-1283;  Hodgetts, R.B., and R.J. Konopka 1973, Genetics 79: 45-54;  Pérez,  M., N. Castillo-Marín, and L.A. Quesada-Allué 1997, Dros. Inf. Serv. 80: 39-41;  Hovemann, B.T., R.P. Ryseck, U. Walldorf, K.F. Stortkuhl, I.D. Dietzel, and E. Dessen 1998, Gene 221: 1-9;  Adams, M.D., et al., 2000, Science 287 (5461): 2185-2195;  Wappner, P., T.L. Hopkins, K.J. Kramer, J.L. Cladera, F. Manso, and L.A. Quesada-Allué 1996a, J. Insect Physiol. 42: 455-461;  Wappner, P., K.J. Kramer, F. Manso, T.L. Hopkins, and L.A. Quesada-Allué 1996b, Insect. Biochem. Molec. Biol. 26: 585-592;  Sambrook, J., E.F. Fritsch, and T. Maniatis 1989, Molecular Cloning, a Laboratory Manual. 2^nd ed. Cold Spring Harbor Laboratory Press, New York.