Bio-Histochemical Analysis of Gene Action in Flimsy Cocoon Mutant of Bombyx mori

The flimsy cocoon mutant of the silkworm, Bombyx mori, is characterized by its thin-layered cocoon-shell. This character is controlled by a recessive gene (referred to as flc) located at 49.0 position on the 3rd chromosome. The investigation of the pattern of action of the flc gene was mainly carried out biochemically and histochemically. Materials used are larvae and cocoons homozygous for flc of the strain b-60 from Institute of Silkworm Genetics, Kyushu University. As a control, the normal silkworms heterozygous for flc are also used. Results obtained are as follows: 1. Flimsy cocoon mutant larva grew as good as the normal and the body weight of flc larva was also similar to that of the normal. 2. The number of cells of the silkgland in flc larva was the same as that of the normal. 3. On the 1st day of the 5th instar the weight and the shape of the silkgland of flc were the same as those of the normal. But owing to the decrease of gelatinous silk protein secreted into the lumen, the weight of the middle silkgland of flc became much lighter than that of the normal, and the shape of the silkgland of flc deflated after the 3rd day of the 5th instar. 4. The cocoon-shell weight of flc was about one third of the normal and 90% of this decrease was caused by the less amount of fibroin. 5. The electrophoretic analysis of both cocoon-shell protein and gelatinous silk protein as well as the analysis of amino acids of cocoon fibroin were performed. As a result, it is indicated that the flc larva synthesizes the same protein qualitatively as that of the normal. 6. The free amino acid composition in hemolymph of the flc larva was similar to that of the normal to the 3rd day of the 5th instar. Afterwards the 5th day the concentration of specific amino acids, such as glycine and serine, became larger, and the total concentration of amino acids, except for sulfide containing amino acids, also increased as compared with those of the normal. 7. The incorporation of ^<14>C-amino acids into posterior part of the silkgland of flc was less than that of the normal, especially reducing by half on the 5th day of the 5th instar. These results show that the accumulation of a great amount of free amino acids in body fluid of flc is caused by the decrease of incorporation of amino acids into the posterior silkgland. 8. A peak of incorporation of ^<14>C-amino acids into protein of the normal posterior silkgland both in vivo and in vitro was seen on the 4th day of the 5th instar, whereas no peak was recognized in the posterior silkgland of flc and also the quantity of incorporation was distinctly reduced. 9. Both the DNA concentration and the incorporation of ^<3>H-thymidine into DNA of the posterior silkgland of flc were equivalent to those of the normal. Therefore, it is thought that DNA synthesis in the silkgland of flc is almost identical with that of the normal. 10. The RNA concentration of the posterior silkgland of flc was equal to that of the normal to the 3rd day of the 5th instar, but became 75% of the normal during the 4th to the 5th day. On the other hand the incorporation of ^<3>H-uridine into RNA of the posterior silkgland of flc was also equal to that of the normal through the 5th instar. These results suggest that RNA molecules of the posterior silkgland cells of flc are possibly degraded during the 4th to 5th day. 11. The cross section of the posterior silkgland of flc was observed with a light microscope. Afterwards the 3rd or 4th day of the 5th instar, nuclei of the posterior silkgland of fle stop developing and cell organella of the silkgland cell of flc degenerate and fibrous granules are accumulated in the cytoplasm of the fie cell, but not in the normal. There are many vacuoles or vesicles in the cytoplasm of the posterior silkgland cells of matured flc larva, but not in the normal one. From all these observations it is thought that on and after the 4th day of the 5th instar, the posterior silkgland of flc is going to break down. 12. Consequently, the fic mutant character due to a thin-layered cocoon-shell may be considered as the effect of the fic gene on the fibroin synthesis, not on the secreting ability itself, in the posterior silkgland cells.薄紙繭蚕は,第3連関群49.0座位に位置する劣性遺伝子’flc’の支配下にあり,最終的に著しく薄い繭を形成する.このflcホモ型である薄紙繭蚕に現われる遺伝子の作用を,flcヘテロ型である正常蚕と比較して,主として生化学的・組織学的に追求した. 1.薄紙繭蚕の幼虫生育経過・発育に伴う体重推移は正常蚕とほぼ同じであった. 2.絹糸腺の細胞数は,薄紙繭蚕と正常蚕との間で差はなく,絹糸腺重量・外部形態ともに,5令1日目までは,薄紙繭蚕絹糸腺も正常蚕絹糸腺と同様に発達することを示した.5令3日目から熟蚕にかけては,薄紙繭蚕絹糸腺重量が正常蚕より軽くなり,外観的にも液状絹の欠乏状態を示した. 3.薄紙繭蚕の繭層重は正常蚕のそれの約1/3であり,その減少量の90%がフィブロイン量の減少に由来した. 4.繭層蛋白質・液状絹蛋白質両者の電気泳動による分析,及び繭層フィブロインのアミノ酸分析において,薄紙繭蚕の造成する絹蛋白質は正常蚕のそれとほぼ同じ性質であった. 5.薄紙繭蚕の体液遊離アミノ酸組成は5令3日目においては正常蚕と類似したが,5日日以後はグリシン・セリンなどの濃度が高くなり,アミノ酸総量(含硫アミノ酸を除く)も正常蚕より高かった. 6.薄紙繭蚕後部糸腺の放射性アミノ酸とりこみ量は正常蚕に比べて少なく,特に5令5日目には,正常蚕の50%であった. 7.後部糸腺の一定量の蛋白質の放射性アミノ酸とりこみ量は,in vivo, in vitro(ミクロソーム分画)両条件下において,正常蚕では5令4日目頃に高いピークがあるのに対して,薄紙繭蚕では2日目から6日目まで,一定の低いとりこみ量を示した. 8.薄紙繭蚕後部糸腺のDNA量,DNAの^<3>H-チミジンとりこみ量ともに,正常蚕とほぼ同じであった. 9.薄紙繭蚕後部糸腺のRNA量は5令3日目までは正常蚕とほぼ同じであったが,5令4日目及び5日目には正常蚕の約75%であった.RNAへの^<3>H-ウリジンとりこみ量には差がなかったので,4～5日目にはRNA分子が分解されている可能性が考えられる. 10.薄紙繭蚕後部糸腺横断面を光顕的に観察すると,細胞の大きさは正常蚕とほぼ同じであり,細胞内部の状態も5令2日目までは正常蚕と同様であった.3日目以後,核の発達が停滞して,分岐数が正常蚕より少なくなった.5令4日目には,薄紙繭蚕後部糸腺細胞において細胞内小器官の退行とフィブロイン顆粒の異常蓄積が観察され,熟蚕時には正常蚕では見られない大きな液胞ないし空胞が多数観察され,細胞の内部崩壊の様相を呈した. これらの結果から,薄紙繭蚕後部糸腺では5令盛食期にフィブロイン合成を高めるために必要なもの,あるいは,状態に異常があり,そのために5令経過が進むにつれて異常性が拡大されて,細胞の崩壊に到ることが推察される