Identification of proteins and developmental expression of RNAs encoded by the 65A cuticle protein gene cluster in Drosophila melanogaster

https://doi.org/10.1016/S0965-1748(97)00107-0Get rights and content

Abstract

Proteins of the third instar larval cuticle of Drosophila melanogaster, LCP5–LCP9, were purified and their N-terminal sequences determined. Three of these proteins (LCP5, 6, and 8) were found to be encoded by two multicopy genes previously mapped to the gene cluster at 65A 5–6 on the left arm of the third chromosome. The analysis of the patterns of developmental expression of the 8 distinct genes at this site showed that all but two were expressed during larval life. The patterns fell into three groups: one where expression was all through larval life, one where expression was primarily in the third instar, and one only during the production of the adult cuticle. One duplicated gene was not expressed in the Canton S strain at any time from the embryo to adult ecdysis. These findings indicate that there is not a unique set of cuticle proteins in the third versus the first and second instar larval cuticles and indicates that overlapping gene sets in several different gene clusters encode the proteins of the cuticle of different developmental stages.

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,

References (41)

Cited by (25)

  • Cuticular Proteins

    2012, Insect Molecular Biology and Biochemistry
  • Cuticular Proteins

    2011, Insect Molecular Biology and Biochemistry
  • The regulation of expression of insect cuticle protein genes

    2010, Insect Biochemistry and Molecular Biology
    Citation Excerpt :

    Since DHR38 is an activator of ACP65A (Bruey-Sedano et al., 2005; Kozlova et al., 2009), the suppressive effect of Broad is thus mediated, at least in part, by the suppression of DHR38 expression. Consistent with its role as a specifier of the pupal stage, Broad misexpression in the second larval instar suppressed expression of the larval cuticle gene LCP65Ab (Charles et al., 1997, 1998; Zhou and Riddiford, 2002). Conversely, Broad proteins were shown to be activators for pupal CP genes.

  • Drosophila cuticular proteins with the R&R Consensus: Annotation and classification with a new tool for discriminating RR-1 and RR-2 sequences

    2007, Insect Biochemistry and Molecular Biology
    Citation Excerpt :

    Riddiford was involved in another major contribution to insect cuticle biology. Charles et al. (1997, 1998) identified a region at band 65A on the Drosophila melanogaster chromosome 3L that had genes for 12 cuticular proteins of the RR-1 type and a pseudogene. Two pairs of genes were very similar, and their copy number varied among strains.

  • Cuticular Proteins

    2005, Comprehensive Molecular Insect Science
View all citing articles on Scopus
View full text