Identification of proteins and developmental expression of RNAs encoded by the 65A cuticle protein gene cluster in Drosophila melanogaster
Introduction
Insect cuticle is composed of the surface epicuticle which contains proteins but lacks chitin and an inner procuticle made of chitin and proteins. There is usually a large number of distinct proteins in individual cuticles (Roberts and Willis, 1980, Cox and Willis, 1985, Andersen and Højrup, 1987); and of those that have been sequenced, there is a clear distinction between those of flexible and those of stiff cuticles (Andersen et al., 1995, Willis, 1996for reviews). Thus, the understanding of cuticular properties will presumably require the characterization of all its major as well as minor proteins. Cuticle protein genes have been isolated from Diptera, Lepidoptera, and Coleoptera (Andersen et al., 1995, Qiu and Hardin, 1995, Charles et al., 1997) and provide a means of assessing the developmental patterns of appearance of the RNAs encoding these proteins.
The larvae of Drosophila melanogaster have a flexible larval cuticle with ventral sclerotized denticle belts (Martinez Arias, 1993). At the onset of metamorphosis the third instar larval cuticle is transformed into the puparium that protects the developing pupa and adult. Analysis of the urea-soluble proteins of these larval cuticles has shown that the major proteins of the third instar larval cuticle are different from those of the first and second instar larval cuticle (Chihara et al., 1982). The third larval instar cuticle contains only five major, and perhaps five minor proteins (Fristrom et al., 1978). The genes encoding four of the major third instar larval cuticle proteins (LCP1–4) are clustered at site 44D on the polytene chromosomes (Snyder et al., 1981, Snyder et al., 1982), while those for LCP5, LCP6, LCCP8 and LCP10 are not separable by recombination and map to position 11 on the third chromosome (Chihara and Kimbrell, 1986). Recently, we analysed a cluster of 12 cuticle protein genes that maps at 65A (coincident with position 11) on the third chromosome (Charles et al., 1997). We show here that this cluster encodes the LCP5, LCP6 and LCP8 proteins and contains another 8 distinct genes for which there are as yet no identified products. Nine of these genes produce RNAs during larval life, one is expressed only during the formation of the adult cuticle, and two are apparently not expressed at any stage from embryo to adult ecdysis.
Section snippets
Fly stocks
D. melanogaster were grown on standard agar-molasses-cornmeal-yeast media. Three wild-type strains were used: Canton Special (Canton S); Oregon R; and Sevelen, obtained from Dr. G. Schubiger, University of Washington, a "wild-type" strain that was originally collected in Zurich, Switzerland. The iso-1 strain (y[1]; cn[1] bw [1] sp [1]) which is isogenic for all chromosomes (Brizuela et al., 1994) from the Bloomington Stock Center was also used.
Developmental staging
Developmental studies were carried out using Canton
Amino-terminal sequence determination of third instar cuticle proteins
The nondenaturing gel profiles of the third instar cuticle proteins of the strains examined in this study are shown in Fig. 2A. The mobilities for all proteins of Canton S and Oregon R were identical, which is consistent with essentially identical allelic forms of the proteins. The "wild type" Sevelen and the isogenic iso-1 lines had a very faint or absent LCP8 band, and a weak LCP6 band (noted "minor") that migrated somewhat slower than the corresponding Oregon R and Canton S bands. The
Discussion
The third instar cuticle of Drosophila melanogaster contains six major (LCP1–6) and at least four minor (LCP7–10) urea-soluble proteins (Fristrom et al., 1978, Chihara et al., 1982; see also Fig. 2A). LCP1–4 are encoded by four genes clustered within 7.9 kb of genomic DNA that maps to 44D (Snyder et al., 1982). These studies show that LCP5, LCP6, and LCP8 are encoded by at least two of the genes clustered at 65A (Charles et al., 1997) and that whereas LCP5 and LCP6 (Lcp-b) are primarily third
Acknowledgements
We thank Wanda Moats for her help with the Drosophila cultures and Dr. Kiyoshi Hiruma for help with the figures. This work was supported by National Science Foundation grants IBN 9100463 and IBN9419957 to LMR. CJC is particularly indebted to the students who participated in much of the research: Greg Schneider, Sharon Jiang, Padmaja Mandalaparthy, Christian Wade and Mario Pineda. CJC was supported in the early years of this work by a Bristol Myers Squibb Company Grant of Research Corporation,
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