In vivoにおけるカイコフィブロイン遺伝子の基本的なプロモーターの同定と解析

博士論文

The Bombyx mori silk genes provide an excellent model system for studying the control of gene expression in differentiated cells. Fibroin gene is actively transcribed in the posterior silk gland cells and completely suppressed in the middle silk gland cells, while sericin genes are actively transcribed in the middle silk gland cells and completely suppressed in the posterior silk gland ce11s. To understand the underlying regulatory mechanisms of this differential expression in silk glands,the transcriptional signals of these genes, especlally of the fibroin gene, have been studied with in vitro transcription systems. These studies have revealed that the transcriptional promoter of the fibroin gene is on the sequence from -29 to +6 (+1 corresponds to the mRNA initiation site), and more detailed analyses using a series of single base substitution mutants have shown the importance of the nucleotides in the TATA box and in the -20 region for the in vitro initiation. However, for the dissection of the eucaryotic promoter, in vitro transcription systems could have intrinslc limitations.Some factors could be lost from the whole cell extracts or inactivated during preparation steps. Proteins tightly bound to the nuclear membrane or lncluded in complex chromatin structure might be subjected to such possibilitis. Furthermore, in eucaryotic cells DNA is packaged in nucleosomes to form chromatin, which is a completely different situation from the in vitro systems. In this paper we describe the ldentifIication of the DNA sequences necessary for faithful and efficient initiation of transcription of the fibroin gene in a living cell system. For this purpose we used a transient expression system consisting of monkey COS cells and a SV40 origin vector. COS cells possess the gene for SV40 T antigen on their chromosomes and constitutively produce that protein. Plasmids that have SV40 origin sequence are replicated in COS cells when introduced into the cell nuclei. We have constructed a fibroin-SV40 origin　plasmid, pSVOFb, and transfected it with fine calcium phosphate precipitates into COS cells. About 48 hours after the DNA transfection, total RNA was extracted and analyzed with nuclease Sl mapping and primer extension technique to see whether faithful transcripts of the fibroin gene were produced in those cells. By either method we have detected fibroin transcripts initiated at the same point as the authentic fibroin mRNA that was obtained from the silk gland. In addition, these transcripts were polyadenylated indicating that the signal for polyadenylation on the fibroin gene was recognized in the COS cells. To study the DNA sequences responsible for this faithful transcription of fibroin gene, a series of 5' deletion genes had been artificially constructed by recombinant DNA technology and their template activities were examined. Deletion genes 5' A -86, -115, -73 and -44 all showed essentially equal activity for the expression, while a mutant 5'Δ -19 dld not support the faithful transcription (see Fig. 8). We have further tested a deletion gene Δ［-58～-20] that have 13 base pair linker stretch in place of the natural sequence between nucleotide position -58 and -20.This internal deletion gene also gave no faithful transcript. These results have revealed that all the signals responsible for the faithful transcription of the fibroin gene were included in the very proximal sequences that are no farther than -44. The sequence from nucleotide position -44 to -19,which harbors the TATA box, is indispensable for the accurate initiation of the fibrion gene in the COS cells. The immediate upstream region of the fibroin gene was further characterized with exact substitution genes including a series of point mutants (see Fig. 10). The faithful transcription initiation of the fibroin gene was strongly suppressed by an o1igonucleotide block substitution over the TATA box (sb A). Single base substitutions at nucleotide -30, -29 or -28 of the TATA box showed deleterious effects on transcription. The results have unequivocally shown the importance of the individual nucleotides in the TATA box for the faithful transcription of the fibroin gene in vivo. Besides the above substitutions. a block substituion around the cap site (sb B) completely abolished the faithful transcription. Point mutations at nucleotide -10, -7, -4 or +1 also lowered the activity, showing the strict sequence requirement of the cap region. as of the TATA box, for the accurate transcription of the fibroin gene in vivo. Most of studies on eucaryotic promoters in living cell systems have shown that some upstream sequences beyond the TATA box are required for faithful transcription in vivo. In contrast to those results, we have shown that only the TATA box and the cap site region are required for faithful and efficient initiation of the fibroin gene in COS cells. Our studies have defined and characterized the basic ptomoter region fibroin gene.

名古屋大学博士学位論文 学位の種類 : 理学博士(課程) 学位授与年月日 : 昭和59年11月2日