Two novel cry-type gene homologues, tentatively named cryPp1 and cryPp2, were detected in the genomic DNA of Paenibacillus popilliae ATCC 14706T, both of which were similar to genes encoding Cry18 proteins, suggesting their similar modes of action. Ours is the first report concerning multiple Cry homologues, detected from a sole strain of P. popilliae. In order to characterize the roles of the two Cry homologues in pathogenicity, a trial for the expression of recombinant proteins in Escherichia coli was conducted, which revealed that the opti- mization of rare codons, such as supplementation of tRNA genes, was necessary.
Many lethal mutations of the silkworm egg have been reported. The lethal phenotype is exhibited at several stages including blastokinesis, head pigmentation, and body pigmentation. We recently isolated a novel lethal mutant, which the skin color of the mature embryo became a light orange and the embryo died before hatching. In this study, we performed morphological observation and genetic analysis of this lethal mutation. Phenotypic observation showed that the phenotype of this lethal mutant is quite similar to that of the sex-linked chocolate lethal (schl), chocolate (ch), chocolate 2 (ch-2), and maternal chocolate (cm) mutants. We first performed the crossing experiment, which revealed that this mutation is regulated by a single recessive gene, and is not linked to the sex chromosome on which the schl mutation is located. We next performed the pre-linkage test with this lethal mutation and three other mutations, which revealed that an allelic relationship among these mutations could not be detected. Therefore, we determined that this is a novel lethal mutant, which we named light orange lethal (l-og). We next performed the linkage analysis of the l-og mutation using 20 F2 individuals and primer sets designed for each chromosome. The linkage analysis revealed that the l-og mutation is definitely located on chromosome 10. Furthermore, the rough mapping using 119 F2 individuals and 7 primer sets designed for chromosome 10 revealed that the l-og-linked region was narrowed down to approximately 3.7 Mb long on chromosome 10.
Advanced lepidopteran males produce two types of sperm; nucleated eusperm called eupyrene sperm, and anucleated parasperm called apyrene sperm. Apyrene sperm is necessary for successful eupyrene sperm fertilization, and apyrene dysfunction results in sterility. In a previous study, we generated Bombyx mori Sex-lethal (Bm-Sxl) mutants, a phenotype mediated by transcription activator-like effector nuclease (TALEN), and found that the Bm-Sxl gene is essential for apyrene spermatogenesis. One of the Bm-Sxl mutant strains, SxlIn3-homo (SxlIn3/SxlIn3), possessed a 3-bp insertion in the highly conserved RNA binding domain. The sterility of SxlIn3-homo can be rescued by administration of apyrene sperm from triploid males with defective eupyrene sperm. The Bm-Sxl mutants can be maintained using the triploid. However, the production of triploid males is labor-intensive. Considering the logistical challenges of that method, we developed an alternative double copulation method to maintain the mutant using a diploid strain carrying a dominant larval phenotype to preempt the challenging and labor-intensive production of triploid silkworms. In this study, we successfully maintained our SxlIn3-homo strain by double copulation of the female with a striped (pS/pS) and a SxlIn3-homo male.