Organization of the Chorion Genes of Bombyx Mori, of Three Gene Clusters a Multigene Family. 111. Detailed Marker Composition

The chorion patterns produced by progeny from crosses that were used to define the linked gene clusters, C h 1, C h 2 and C h 3 (strain C108 us. strain Ascoli) , were examined by two-dimensional gel electrophoresis (isoelectric focusing us. SDS/urea). Approximately 60 proteins were assigned to four previously defined chorion classes, A, B, C and H,, believed to represent the products of related genes, on the basis of size and relative cysteine content. All strain-specific markers showed co-dominance in the F,, indicating the likelihood that they are not products of post-translational modification. Twentyseven Ascoli markers co-segregated in testcross progeny, and none of the resolved proteins showed independent assortment, confirming their linkage to chromosome 2. Two-dimensional screening of recombinants demonstrated that all three clusters contain Class A and B markers; H, markers have been found only in Ch 1 and Ch 2; whereas, mapped C markers were confined to C h 3. This indicates that chorion clusters are heterogeneous with respect to the markers they contain. silkmoth chorion consists of 50 to 100 different polypeptides that are T7:nthesized in a highly ordered developmental sequence by the follicular epithelium during the terminal stages of oogenesis (PAUL et al. 1972; NADEL and KAFATOS 1980). Similarities in the sizes, amino acid compositions ( KAFATOS et al. 1977) and primary structure of chorion proteins (REGIER et al. 1978; RODAKIS 1978; KAFATOS et al. 1978; JONES et al. 1979) suggest that they are encoded by several families of related genes. To gain an understanding of both the developmental regulation and the evolution of chorion genes, it is essential to know their detailed chromosomal arrangement. UsiEg electrophoretic variants resolved by isoelectric focusing from inbred strains of Bombyx mori as genetic markers, we previously demonstrated that a large number of chorion genes are linked (GOLDSMITH and BASEHOAR 1978), and form at least three clusters on chromosome 2 (GOLDSMITH and CLERMONTRATTNER 1979). We report here the detailed resolution of the set of mapped chorion markers from testcrosses between strains C108 and Ascoli (GOLDSMITH and CLERMONT-RATTNER 1979) by two-dimensional gel electrophoresis and the ' Present address Department of Zoology, University of Rhode Island, Kingston, Rhode Island 02881. Genetics 96: 201-212 September, 1980 202 M. R. GOLDSMITH A N D E. CLERMONT-RATTNER assignment of these markers to four defined protein classes. Our results confirm earlier findings of genetic co-dominance and indicate that chorion gene clusters are heterogeneous with respect to the classes of markers they contain. MATERIALS A N D METHODS Silkworm stocks and rearing: Strains C108 and Ascoli were obtained from Y. TAZIMA and A. MURAKAMI of the National Institute of Genetics, Misima, Japan, and reared on fresh mulberry leaves, as previously reported by GOLDSMITH and BASEHOAR (1978). Solubilization and in vitro labeling of chorion proteins: Chorions were cleaned, solubilized and carboxamidomethylated as described by GOLDSMITH and BASEHOAR (1978). Samples to be labeled with 14CC-iodoacetamide were solubilized at a concentration of 3 to 4 chorions/l0 @1 (approximately 20 pg protein/ pl) in 8 M urea, 0.05 M dithiothreitol, 1.5 mM lysine, 1.5 mM EDTA and 0.05 M Tris-HC1, pH 9.0 at room temperature. To 5 pl of dissolved protein we added 10 p1 of a solution containing 4 M urea, 0.02 M iodoacetamide, 0.6 M Tris pH 8.4 and 2.5-5 @Ci 14C-iodoacetamide (Amersham, 13 to 26 mCi/mM) in subdued light. After 5 to 10 min, a halfvolume of 0.42 M iodoacetamide in 1.2 M Tris-HC1, pH 8.4 was added; the reaction was terminated in 10 to 15 min by the addition of 3 ,pl P-mercaptoethanol per 100 pl final volume. Urea and nonradioactive iodoacetamide were purified as described elsewhere (GOLDSMITH and BASEHOAR 1978). Two-dimensional gel electrophoresis: Samples were resolved by isoelectric focusing, followed by separation in the second dimension on slab gels contain;ng sodium dodecyl sulfate and urea (GOLDSMITH et al. 1979). Mixed samples contained 45 to 60 pg nonradioactive protein and 4 to 8 ug radioactive protein. After electrophoresis, gels were fixed, stained and photographed as reported by GOLDSMITH et al. (1979), soaked in 1 to 3% glycerol for 30 min and dried on Whatman 3 M M filter paper, using a homemade vacuum suction apparatus. Autoradiographic exposures were carried out for 1 to 6 weeks with Kodak X-omat R film.