Molecular cloning of a POU domain transcription factor involved in regulation of Bombyx sericin-1 gene カイコセリシン-1遺伝子の制御に関わるPOUドメインを持つ転写因子のクローニング



    • 福田, 雅一 フクタ, マサカズ



Molecular cloning of a POU domain transcription factor involved in regulation of Bombyx sericin-1 gene




福田, 雅一


フクタ, マサカズ











   The POU domain is a DNA-binding regionconsisting of 75-82<br /> amino acids POU-specific domain, a short variable linker region and<br /> a POU-homeodomain of 60 amino acids (for a review,seeRuvkin and<br /> Finny, 1991). It was originarry found in three mammalian<br /> transcription factors, the pituitary-specific Pit-1/GHF-1,the<br /> ubiquitous Oct-1 and the predominantly B cell specific Oct-2, and<br /> the product of the cell lineage control gene <u>Unc</u>-86 of <u>Caenorhadbitis</u><br /><u>elegans</u> (Herr <u>et</u> <u>al</u>.,1988 and references therein). <br />By means of<br /> sequence similarity, several other mammalian POU domain genes have<br /> also been identified (He <u>et</u> <u>al.</u>, 1989; Monouki <u>et</u> <u>al.</u>, 1990; Okamoto<br /> <u>et</u> <u>al.</u>, 1990; Rosner et al., 1990 Scholer <u>et</u> <u>al.</u>, 1990; Suzuki <u>et</u> <u>al</u>.,<br />1990). All of them were shown to interact with an octamer-like<br /> sequence and to activate transcription <u>via</u> an octamer motif near the<br /> TATA box.The <u>Drosophila</u> Cfl-a protein,which interants with a DNA<br /> element required for expression of the dopa decarboxylase gene in<br /> selected dopaminergic neurons (Johnson and Hirsh,1990), was also<br /> found to posses a POU domain similar to those of the mouse Oct-6<br /> (Suzuki <u>et</u> <u>al</u>.,1990) and the humann Brn-1 and Brn-2 (He <u>et</u> al</u>.,1898)<br />proteins. These POU domain genes are likely regulatory genes<br /> controlling transcription of distinct sets of genes during develop-<br />ment. The finding that two dwarf mutations in mice are null mutations<br /> in the Pit-1/GHF-1 gene (Li <u>et</u> <u>al</u>., 1990) provide further support on<br /> the roles of POU transcription factors in development. Recently, the<br /> mternally expressed POU transcription factor, the Oct-3/4<br /> protein (Okamoto <u>et</u> <u>al</u>., 1990; Scholer <u>et</u> <u>al</u>., 1990), has also been<br /> shown to be required for the first embryonic cell division in mice<br /> (Rosner <u>et</u> <u>al</u>., 1990).<br />   Suzuki and his colleagues have been studying the developmental<br /> regulation of the silk protein genes in <u>Bonbyx</u> <u>mori</u></i> (Suzuki <u>et</u> <u>al</u>.,<br /> 1990). Among them, the siricin-1 gene is expressed exclusively in the<br /> middle silk gland while the fibroin gene is specific to the posterior<br /> silk gland. Both genes are actively expressed during the intermolt<br /> but repressed during the molting stages. Several silk gland protins<br /> have been identified as putative regulatoly factors involved in the<br /> transcriptioal control of the fibroin and scrin-1 genes ( Hui <u>et</u> <u>al</u>.,<br /> 1990; Matsuno <u>et</u> <u>al</u>., 1989,1990). One of these proeins , SGF-3, was<br /> found to bind with high affinity to the SC region of the sericin-1 gene<br /> and the distal upstream reion of the fibroin gene (Hui <u>et</u> <u>al</u>., 1990).<br /> These regions are known to be important for an efficient transcription<br /> in the silk gland extracts. Amultimer of the SC region gave<br /> transcriptional enhancement in extracts prepared from the middle silk<br /> gland where the sericin-1 gene is specifically epressed but that of a<br /> mutant SC region giving a reduced affinity for SGF-3 did not (Matsuno<br /> <u>et</u> <u>al</u>., 1990). Mobility shift assay revealed that SGF-3 is far more<br /> abundant in the silk gland of the 2-day-old fifth-instar larvae than in<br /> the pasterior silk gland (Hui <u>et</u> <u>al</u>., 1990; Matsuno <u>et</u> <u>al</u>., 1990; Y.<br /> Suzuki, unpublised). These obaervations suggest that the SGF-3 is a key<br /> regulatory factor in the transcription control ol the sericin-1 gene. <br />   The SGF-3 was supposed as an octamer binding protein (Hui <u>et</u> <u>al</u>.,<br /> 1990). Since high affnitiy SGF-3 binding sites, such as the SC and<br /> fibroin distal upstream regions, also contain octamer-like sequences,<br /> it has been speculated that SGF-3 might possess a POU domain similar<br /> to the mammalian octamer-binding proteins. This repoort presents here<br /> the isolation and characterization of a POU domain containg cDNA<br /> (POU-M1) from the middle silk gland. It encodes a protein with a POU<br /> domain identical to that of the <u>Drosophila</u> <u>Cfl-a</u> protein. By mobility<br /> shift assay and nuclease protection assay, the POU-M1 protein and the<br /> putative silk gland factor SGF-3 were found to interact in an<br /> indistinguishable manner with the SC region of the sericin-1 gene,<br /> which is a key <u>cis</u>-acting element involved in the stimulation of<br /> sercin-1 gene transcription through the interaction with SGF-3.<br /> Antibodies raised against the synthetic oligopeptides correponding<br /> to the two regions of putative POU-M1 protein reacted specifically to<br /> both the POU-M1 protein and SGF-3. Northern blot hybridization<br /> and Western blotting revealed that the POU-M1 expression is regulated<br /> both temporally and spatially during the silk gland development. It<br /> is concluded that the POU-M1 proein is identical to the SGF-3 and<br /> proposed that the differential expression of the POU-M1 gene is probably<br /> involved in the transcriptional regulation of the silk protein genes.


  1. Contents / p2 (0003.jp2)
  2. 1. Abstract / p3 (0004.jp2)
  3. 2. Introduction / p4 (0005.jp2)
  4. 3. Matreials and Methods / p7 (0008.jp2)
  5. Cloning and Sequencing of POU-M1 cDNA / p7 (0008.jp2)
  6. In vitro Transcription/Translation / p8 (0009.jp2)
  7. Mobility Shift and DNaseI Footprinting Assay / p8 (0009.jp2)
  8. RNA Extraction and Blot-Hybridization (Northern) Analysis / p9 (0010.jp2)
  9. Synthetic Peptides and Immunization / p9 (0010.jp2)
  10. Hyper-shift Assay / p9 (0010.jp2)
  11. Western Blotting / p10 (0011.jp2)
  12. 4. Results / p11 (0012.jp2)
  13. Isolation and Sequence Analysis of POU-M1 cDNA / p11 (0012.jp2)
  14. The POU-M1 Protein and SGF-3 Bind Indistinguishably to the SC Region / p12 (0013.jp2)
  15. Antibodies Raised Against the Deduced POU-M1 Amino Acid Sequence Also Reacted to SGF-3 with the Same Specificity / p14 (0015.jp2)
  16. Developmentally Regulated Expression of the POU-M1 Gene in the Silk Gland / p15 (0016.jp2)
  17. 5. Discussion / p17 (0018.jp2)
  18. 6. Acknowledgements / p22 (0023.jp2)
  19. 7. References / p23 (0024.jp2)
  20. 8. Figures / p29 (0030.jp2)
  21. 9. List of publication / p42 (0056.jp2)


    • 8000000090837
  • DOI(NDL)
  • 本文言語コード
    • eng
  • NDL書誌ID
    • 000000254930
  • データ提供元
    • 機関リポジトリ
    • NDLデジタルコレクション