Genetic analysis of the Nd-s mutation in the silkworm,

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1 Jpn. J. Genet. (1984) 59, pp Genetic analysis of the Nd-s mutation in the silkworm, Bombyx mori BY Fusaho TAKEI, Ken-ichi KIMURA, Shigeki MIZUNo, Toshio YAMAMOTO1' and Kensuke SHIMURA2' Laboratory of Biochemistry, Department of Agricultural Chemistry, Tohoku University, Sendai Miyagi 980 (Received April 27, 1984) ABSTRACT To investigate the distance between Nd-s and Fib-L on the 14th chromosome of Bombyx mori, a three-point test cross involving U, Nd-s, and Fib-L was carried out. The crossover frequency between U and Fib-L or U and Nd-s was 24.6%. Crossingover between Fib-L and Nd-s was not observed. These results indicate that Fib-L is closely linked with Nd-s. In the fibroin slightly secreted into the lumen of the posterior silk gland of the Nd-s mutant silkworm, fibroin L-chain was not detectable. A possibility that the Nd-s phenotype is caused by a mutation in the L-chain gene, is discussed. 1. INTRODUCTION The posterior silk gland cells of the silkworm, Bombyx mori, synthesize a large amount of fibroin in the fifth instar. Fibroin is composed of one H-chain (MW 350,000) and one L-chain (MW 25,000), which are connected by disulfide bonds (Sasaki and Noda 1973; Gamo et al. 1977; Shimura et al. 1982). Genetic analysis by Hyodo et al. (1980, 1984) revealed that the H-chain gene (Fib-H) is linked with the Nd locus on the 25th chromosome3' and the L-chain gene (Fib-L) is linked with the U locus on the 14th chromosome. The Nd-s locus has been shown to be located on the 14th chromosome (Doira 1978). The Nd-s phenotype is characterized by very poor development of the posterior silk gland and very low level production of fibroin (Watanabe 1959). Suzuki et al. (1974) showed that in a heterozygous combination, Nd-s/+, an intermediate level of the H-chain mrna was present, which suggests that the Nd-s mutation behaves codominantly in the heterozygoue. In this communication, we report that Nd-s is closely linked with Fib-L and discuss a possibility that the Nd-s phenotype is actually caused by a mutation in the fibroin L-chain gene. 1) Department of Silkworm Breeding, The Sericultural Experiment Station, Kobuchizawa, Yamanashi, > Department of Biological Scienceș Tohoku Fukushi University, Sendai, Miyagi ) Fib-H was originally assigned to the 23rd chromosome because of its linkage to Nd (Hyodo et al. 1980). The assignment was changed according to the recent notion that the correct locus for Nd is on the 25th chromosome (Doira 1983).

2 308 F. TAKEI, K. KIMURA, S. MIZUNO, T. YAMAMOTO and K. SHIMURA Stocks 2. MATERIALS AND METHODS Three stocks of Bombyx mori, U Nd-s, + +Fib-LA (J-131 strain), + +Fib- LC (Tamanashikasuri) were used for the present experiment. The loci U, Nd-s and Fib-L, are all located on the 14th chromosome (Doira 1978). Map units for U and Nd-s have been determined to be approximately 19.2 and 40.5, respectively (Doira 1978). The U phenotype is characterized by dark brown pigments covering dorsal and lateral sides of the body (Doira 1978). Fib-LA and Fib-LC are distinguishable from the electrophoresic mobility of the fibroin L-chain (Hyodo et al. 1984). Mating If an F1 male (U Nd-s Fib-L / + + Fib-LC) is mated with a female (+ + Fib-LC), Fib-L /Fib-LC and Fib-LC/Fib-LC combinations in the F2 are indistinguishable, because both of these F2 types show only C type as the L-chain phenotype. Fib-L is characterized by the absence of L-chain in the fibroin secreted into the lumen of the posterior silk gland (see RESULTS AND DIS- CUSSION). In order to distinguish Fib-LC and Fib-L in the F2, an F1 male (U Nd-s Fib-L /-F + Fib-C) was mated with a female (+ + Fib-LA). In this combination, Fib-L /Fib-LA shows A type and Fib-LC/Fib-LA shows AC type, respectively. Preparation of fibroin secreted into the lumen of the posterior silk gland Posterior silk glands of the silkworm at the fifth day of the fifth instar were excised, washed briefly in cold 1.15% KCl and immersed immediately in 60% ethanol. After standing in the cold overnight, the silk glands were ground in a mortar in 60% ethanol. The fibroin secreted into the lumen was coagulated under these conditions and gave string-shape and glassy look, which was separated easily from the tissue proteins with a pair of forceps. Fibroin thus obtained was washed several times with 90% ethanol, once with ether and dried in a desiccator. Electrophoresis of fibroin subunits Fibroin or cocoon was dissolved in 60% LiSCN, dialyzed against O.O1M sodium phosphate buffer (ph 7.5) containing 1 % sodium dodecyl sulfate (SDS) and 5M urea and subjected to SDS-12.5% polyacrylamide gel electrophoresis, according to the method of Laemmli (1970). Sericin in the cocoon did not interf er with the determination of the mobility of fibroin L-chain.

3 Southern Close linkage of Nd-s and Fib-L in Bombyx mori 309 blot hybridization DNA was extracted from the posterior silk glands as described by Manning and Gage (1978). Restriction endonuclease digestion was carried out under the conditions recommended by the suppliers (TAKARA Shuzo Co. LTD, Kyoto). Digested DNA fragments were fractionated by horizontal 0.7% agarose gel electrophoresis, transferred from gels to nitrocellulose filters, and hybridized with the 32P-labeled L-chain cdna clone pla23 (Kimura et al. in preparation), which had been labeled with [a-32p] dctp (Amersham) by nicktranslation. The filter hybridization was carried out as described by Denhardt (1966). After hybridization, the filter was washed, dried and exposed to Kodak Ortho G film at -70 C. 3. RESULTS AND DISCUSSION Recently we have found that phenotypes of fibroin L-chain can be classified into at least three groups (A, B and C types) from its mobility on the SDS- 12.5% polyacrylamide gel electrophoresis (Hyodo et al.1984). To determine the type of L-chain produced by a mutant silkworm (U Nd-s), the fibroin secreted slightly into the lumen of the posterior silk gland was subjected to SDS-12.5 % polyacrylamide gel electrophoresis after reduction with 2-mercaptoethanol. However, L-chain was not detectable (Fig. 1, lane 1). When fibroins from hybrid silkworms, U Nd-s J + + (Tamanashikasuri) and U Nd-sl + + (J-131) were examined, the phenotype of L-chain was only C type derived from the Tamanashikasuri allele (Fib-LC) in the former and only A type from the J-131 allele (Fib-LA) in the latter (Fig. 1, lanes 2 and 3). From these results, it is evident that the L-chain from the U Nd-s allele is not present in a detectable amount in the fibroin secreted into the lumen of U Nd-sl U Nd-s or U Nd-s/ + +, accordingly we classified the L-chain gene in the U Nd-s strain as 0 type (Fib-L ). To investigate whether an L-chain gene exists in the U Nd-s strain, genomic DNA extracted from the posterior silk gland of the U Nd-s strain or Tamanashikasuri was digested with EcoRI and subjected to Southern blot hybridization with 32P-labeled L-chain cdna clone pla23. Fig. 2 shows that both genomic DNAs derived from U Nd-s and Tamanashikasuri produced one main band of hybridization at 5.0 kbp. These results indicate that the phenotype of Fib-L is not caused by complete deletion of the L-chain gene. Genetic distance between the Fib-L locus and the Nd-s locus was measured by the backcross + + Fib-LA X (U Nd-s Fib-L / + + Fib-LC). Fib-L phenotypes of offsprings were examined by SDS-12.5 % polyacrylamide gel electrophoresis of the cocoon proteins after reduction with 2-mercaptoethanol. Numbers of offsprings showing crossed-over phenotypes or non-crossed-over phenotypes with respect to U, Nd-s, and Fib-L are summarized in Table 1.

4 310 F. TAKEI, K. KIMURA, S. MIZUNO, T. YAMAMOTO and K. SHIMURA Fig. 1. Comparison of fibroin L-chains secreted by different strains. Fibroin secreted was subjected to SDS-12.5% polyacrylamide gel electrophoresis after reduction with 2-mercaptoethanol and stained with Coomassie brilliant blue R. Lane 1; U Nd-s, lane 2; U Nd-s/+ + (Tamanashikasuri), lane 3; U Nd-s/+ + (J-131), lane 4; a mixture of U Nd-s/+ + (Tamana.shikasuri) and U Nd-s/+ + (J-131). An enlarged photograph for the L-chain region is shown in the lower panel. Fig. 3 shows the types of L-chain in the offsprings showing the phenotype of U Nd-s, + +, U+, or +Nd-s. Four types of offsprings; i.e. U Nd-s AC, + +A, U + A, + Nd-s AC, were not found, indicating that the crossing over between Nd-s and Fib-L did not happen. A crossing-over value between U and Nd-s, or U and Fib-L, was 24.6% (= ( )/492). From these results, we conclude that Fib-L and Nd-s are closely linked. It is conceivable that the Nd-s locus is included in the L-chain gene itself or in its flanking regions.

5 Close linkage of Nd-s and Fib-L in Bombyx mori 311 Fig. 2. Detection of the -chain gene sequence in the genomic DNA. DNA prepared from the posterior silk gland was digested with EcoRI and subjected to Southern blot hybridization with 32P-labeled L-chain cdna clone pla23, lane 1; U Nd-s, lane 2; + + (Tamanashikasuri). Table 1. Phenotypes of ofsprings from the backcross+ +Fib-Lx (U Nd-s Fib-L /+ +Fib-LC) As shown in Fig. 1, L-chain is not detectable in the fibroin secreted slightly into the lumen of the posterior silk gland of U Nd-s (lane 1). Fbbroin secreted by the hybrid (U Nd-s/ + +) contained L-chain from the + (Tamanashikasuri or J-131) allele only (lanes 2 and 3). However, we have noticed recently that immunologically detectable L-chain is present in the posterior silk gland tissue of the U Nd-s strain (unpublished observation). Thus, it is likely that the L-

6 312 F. TAKEI, K. KIMURA, S. MIZUNO, T. YAMAMOTO and K. SHIMURA Fig. 3. Electrophoretic profiles of the L-chain produced by offsprings of different phenotype. Offsprings from the backcross++fib-la x (U Nd-s Fib-L0/++Fib-LC) were classified into four phenotype (U Nd-s, + +, U+, +Nd-s). Ten individuals were selected from each group and each cocoon protein was subjected to SDS-12.5% polyacrylamide gel electrophoresis after reduction with 2-mercaptoethanol and stained with Coomassie brilliant blue R. chain synthesized in the U Nd-s strain can not combine with the H-chain and remained within the posterior silk gland cells. Recently we have postulated that the H-L subunit structure of fibroin is important for the normal level secretein (Takei et al. 1984). The Nd-s mutant seems to be a good system for studying further on the role of L-chain

7 Close linkage of Nd-s and Fib-L in Bombyx mori 313 in the synthesis and secretion of fibroin. This work was supported by a Grant-in-Aid for Scientific Research No from the Ministry of Education, Science and Culture, Japan. REFERENCES DENHARDT, D. T. (1966) A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, DOIRA, H. (1978) Genetic stocks of the silkworm. In The Silkworm: An Important Laboratory Tool (ed. Y. Tazima), pp Kodansha, Tokyo. DOIRA, H. (1983) Linkage maps of Bombyx mori-status quo in Sericologia 23, GAMO, T., INOKUCHI, T. and LAUFER, H. (1977) Polypeptides of fibroin and sericine secreted from the different section of the silk gland in Bombyx mori. Insect Biochem. 7, HYoDo, A., GAMO, T. and SHIMURA, K. (1980) Linkage analysis of the fibroin gene in the silkworm, Bombyx mori. Jpn. J. Genet. 55, HYODO, A., YAMAMOTO, T., VEDA, H., TAKEI, F., KIMURA, K. and SHIMURA, K. (1984) Linkage analysis of the fibroin light chain gene in the silkworm, Bombyx mori. Jpn. J. Genet. 59, LAEMMLI, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London). 227, MANNING, R. F. and GAGE, L. P. (1978) Physical map of the Bombyx mori DNA containing the gene for silk fibroin. J. Biol. Chem. 253, SASAKI, T. and NoDA, H. (1973) Studies on fibroin of Bombyx mori directly extracted from the silkworm. Biochim. Biophys. Acta. 310, SHIMURA, K., KIKUCHI, A., KATAGATA, Y. and OHOTOMO, K. (1982) The occurrence of small component proteins in the cocoon fibroin of Bombyx mori. J. Sericult. Sci. Jpn. 51, SUZUKI, Y. and SUzuKI, E. (1974) Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori. J. Mol. Bio. 88, TAKEI, F., OYAMA, F., KIMURA, K., HYODO, A., MIZUNO, S. and SHIMURA, K. (1984) Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd (2). J. Cell Biol. in press. WATANABE, T. (1959) Studies of the sericin cocoon, (1) Chemical properties of the domestic silkworm spinning sericin cocoon. J. Sericult. Sci. Jpn. 28,