Many thousands of recombinant proteins have been successfully produced in baculovirus - infected insect cells and larvae. In this study, to improve its value and the yield of recombinant protein production, we constructed transgenic silkworm using Heat shock genes with regard to protein folding. This time, we adapted GAL4/UAS system to express at necessary time point and to carry genes for foreign protein. First, we generated two transgenic cells and silkworm lines that carried the silkworm heat shock proteins, UAS-HOP and UAS-HSC70 and UAS-HSP70 and UAS-HSP40 construct plus 3xP3-DsRED. Subsequently, to drive the GAL4 gene as activatorvector, we engineered Baculoviruses that contain the GAL4 under the P10 promoter linked to the expression cassette of interest foreign genes under the polyhedron promoter. Also, activator vector linked to the GAL4 was designed expressing 6xHis and 6xHis–GST tag. Infection of silkworm larvae with recombinant virus, His-tagged human C3d gene was more efficiently produced transgenic silkworm than that of wild-type, but not His-GST tagged. We show the possibilityin use of HSPs transgenic silkworm system by GAL4/ UAS BmNPV that can generate the efficient production of foreign protein.

The genomic structure and phylogenetic relationships of HSP88 genes from P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa are described. The HSP88 genomic DNA from P. tenuipes Jocheon-1, P. tenuipes and C. militaris all contain 5 introns and 6 exons with the length of 13, 62, 32, 1438, 306, 288 bp, encoding 713 amino acid residues. C. pruinosa HSP88 genomic DNA contains 4 introns and 5 exons encoding 713 amino acids. The length of each exon of C. pruinosa HSP88 is 13, 62, 32, 1744, 288 bp and the length of exon 4 is identical to the total length of exon 4 and exon 5 of HSP88 of P. tenuipes Jochoen-1, P. tenuipes, and C. militaris. The deduced amino acid sequence of P. tenuipes Jocheon-1 HSP88 showed 99% identity with the P. tenuipes, 97% identity with the Cordyceps militaris, and 98% identity with the C. pruinosa. Phylogenetic analysis confirmed that the P. tenuipes Jocheon-1, P. tenuipes, C. militaris and C. pruinosa HSP88 are placed together within the ascomycetes group of fungal clade.

In this study, a full-length heat shock protein88 complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening of P. tenuipesJocheon-1 Uni-Zap cDNA library and 5' RACE polymerase chain reaction. The Paecilomyces tenuipes Jocheon-1 heat shock protein88 cDNA contains an open reading frame of 2,139 bp encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipes Jocheon-1 HSP88 cDNA showed 77% identity to N. haematococca HSP88 and 45-76% identity to other fungi HSP88. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade and P. tenuipes Jocheon-1 HSP88 also contains the conserved ATPase domain at the N-terminal. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as a 88 kDa polypeptide in baculovirus-infected insect Sf9 cells. Under different stress conditions, mRNA expression of P. tenuipes Jocheon-1 HSP88 were quantified by real-time PCR and the result showed that heat shock stress affected the mRNA expression levels of P. tenuipes Jocheon-1 HSP88.

For stable germline transformation, the promoter of B. mori cytoplasmic actin gene (BmA3) was used to ubiquitous expression of transgenes. Except for BmA3 promoter, promoters used to regulate gene expressionin all tissues and developmental stages of B. mori were not nearly developed. To identify more powerful promoter than previously reported BmA3 promoter (Mange et al., 1997), we introduced a new dot blot hybridization method, and isolated nine clones that show stronger dot signal compared to the control, BmA3by this method. Among these 9 clones, we focused on one clone which has high amino acid homology (94%) with heat shock protein 70 gene of Trichoplusia ni. This resulting positive clone, named bHsp70 (B. mori heat shock protein 70) was ubiquitiously expressed in tissues and developmental stage of fifth instar B. mori larvae,and stimulated bythermal and ER stress. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1003/+147) in the 5'-flanking region of bHsp70 gene that has 264-fold more intensive promoter activity than BmA3 promoter. Moreover, transcription activity of bHsp70 promoter under heat shock condition (42 ℃, 4 hr) was increased over 2-fold than normal condition. Therefore, we suggest that bHsp70 promoter may be used more effective candidate for transgene expression in B. mori.

We investigated the effects of cadmium exposure and various stress on the transcription of heat shock protein 70 and 82 (HSP70 and HSP82) from Pardosa astrigera wolf spider. To do this, P. astrigera HSP70 and HSP82 genes were cloned and its full-length sequence determined. Female spiders were long-term exposed to cadmium or to polychlorinated biphenyl (PCB) for 2, 4 and 6 weeks and short-term exposed to endosulfan by dietary uptake. Female spiders were also exposed to various temperatures. HSP82 did not show a clear tendency of transcription induction following exposure to cadmium. On the contrary, HSP70 transcription gradually increased during the exposure to 2, 20 and 40 mM of cadmium for 2, 4 and 6 weeks. Transcript level of HSP70 was not significantly changed by endosulfan and PCB exposure. In the short-term (3 hr) temperature exposure, an increased expression of HSP70 was observed under the heat shock to 30°C and then slightly decreased at 35°C. However, induction of HSP70 transcription was not observed during the long-term (7 days) temperature exposure. Taken together, HSP70 gene appears to be up-regulated by cadmium in a time-dependent manner but little affected by other potential contaminants. Analysis of HSP70 transcript levels in P. astrigera collected from various fields revealed that levels of cadmium concentration were well correlated with HSP70 transcript levels (r2 = 0.76). Taken together, it was suggested that transcript level of HSP70 could be useful as a biomarker for the long-term cadmium exposure of P. astrigera.

Recently Transgenesis was achieved in Bombix mori. For stable and effective transgenesis in B.mori, B.mori cytoplasmic actin gene (BmA3) promoter was used to expression of marker gene, the green fluorescent protein(GFP). Green fluorescent protein expression for selection of transformants was visible in all larval, pupal, and adult tissues but, unexpectdly, was not detectable in embryos. So, it spend times and money on rearing of silkworm. Furthermore, the BmA3 promoter is predominantly active in the midgut, which makes it difficult to reliably identify transformants since autofluorescence of many insect foods can mask low-level fluorescence and only allows the detection of strongly expressing individuals with potentially multiple insertions. Therefore, we need more intensely promoter than BmA3 promoter for selected by expression of GFP in embryos and selected by reliable expression of GFP in larvae. We performed dot blot hybridization to develop strong promoter. Nine differentially expressed clones were isolated and we focused one clone of them which has high similarity with heat shock protein 70 gene from D.melanogaster. We named it as bHSP70 (Bombyx mori heat shock protein 70). Expression from the hsp70 promoter was strong and heat shock-dependent. And Drosophila hsp70 promoter appears useful for regulating expression of Exogenous DNA. So, we analyzed transcriptional activity of promoter with bHSP70 gene by using dual luciferase assay system. bHSP70 promoter has about 264 folds more intensely than BmA3 promoter. Also, when bHSP70 promoter treated heat shock(42℃), transcriptional activity incresed 2 times more than normal condition. Therefore, we suggest that bHSP70 promoter is more effective candidate for stable transformation and selection of transformants.

We examined the effects of cadmium exposure and various temperature stress on the expression of Pardosa astrigera heat shock protein 70 (HSP70). To do this, P. astrigera HSP70 gene was cloned and its sequence determined. Female spiders collected from non-contaminated region were exposed to 40mM CdCl2 for 2, 4 and 6 weeks by dietary uptake. At the end of every 2, 4 and 6 weeks of exposure, a batch of 5 spiders was collected and total RNA was extracted from each batch of whole bodies. Female spiders were also exposed to different temperatures (20, 25, 30 and 35℃) for 3h and RNA extracted likewise. Transcription profiles of HSP70 in response to cadmium and temperature were determined by quantitative real-time PCR using 18S rRNA as reference gene for data normalization. HSP70 transcription gradually increased during 2,4 and 6 weeks of exposure to cadmium. In particular, the expression level at 6-week exposure was 3.4-fold higher than that of untreated control. In the temperature response, an increased expression of HSP70 was also observed as temperature increased up to 30℃ and then slightly decreased at 35℃. The expression level at 30℃ was 2.3-fold higher than that of 25℃. Taken together, HSP70 gene appears to be up-regulated by general stress factors including cadmium exposure and temperature increase.

외부에서 투여된 열자극, 알콜 및 생리적 염과 같은 환경 스트레스는 체내 각 부위에서 스트레스단백질(열자극단백질, HSP)을 생성하게 된다. 본 연구에서는 비소가 흰쥐 대동맥의 수축에 미치는 영향을 조사하기 위해 스트레스단백질의 발현과 대동맥의 수축력의 변화와 이들과의 관계를 알아보고자 실험을 실시하였다 적출한 혈관은 organ bath에 담가 0, 0.5, 1, 2,및 4 mM As를 처리한 후 1, 3, 및 8시간 뒤에 KCI(55 mM)에 대한 수

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

엽록제 small HSP의 기능을 조사하기 위하여 항상적으로 발현하는 형질전환 식물체를 작성하였다. 고온 스트레스 하에서의 형질전환 식물체의 고온내성을 chlorophyll 형광으로 측정하였다. Leaf disc를 고온조건에서 5분간 처리한 후, 광화학계 II의 불활성화를 나타내는 Fo 값의 증가 또는 Fv 값의 감소치를 조사하였다. 형질전환 식물체는 고온 스트레스 하에서의 이들 값의 증감율이 현격히 감소하였다. 또한 유식물체를 에서 45분간 처리한 후, 에서 계속적으로 배양하였을 때, 비형질전환 식물체는 전부 고사하였으나, 형질전환 식물체의 약 80%는 생존하였다. 이러한 결과는 엽록체 small HSP가 고온 스트레스 하에서 광합성기구를 보호하는데 있어서 중요한 기능을 담당하고 있음을 나타낸다.

고등식물에 있어서 엽록체에 존재하는 저 분자량 heat shock protein (smHSP)은 식물의 내열성 획득에 있어서 필수유전자임이 mutant를 이용한 유전학적인 연구에 의해 보고된 바 있다. 고온내성이 강한 작물인 벼로부터 엽록체 smHSP cDNA를 분리하고자 벼의 잎에서 분리한 mRNA로 작성한 cDNA library로부터 screening하였다. 선발된 smHSP cDNA는 1,026 bp의 염기로 구성되어 있었으며, 239개의 아미노산

본 연구는 食品衛生과 環境衛生상 주요 危害因子중의 하나인 카드뮴에 대하여 식품으로서 섭취기회가 많은 마늘성분과 비타민 A를 이용한 危害 경감 혹은 방어효과를 평가하고, 카드뮴暴露에 대한 細胞水準의 反應으로서 HSP(Heat Shock Protein, 熱衝擊蛋白質 혹은 스트레스蛋白質)의 發顯時期와 發顯程度를 관찰하고자 실시하였다. 실험동물로는 Wistar계 SPF 수컷 랫드 483마리를 사용하였으며, 對照群, 카드뮴投與群(Cd, CdCl₂20 ㎎/㎏), 카드뮴과 마늘유 (diallyl disulfide) 投與群(Cd+Dds, diallyl disulfide 50 ㎎/㎏), 카드뮴과 비타민 A(retinol acetate) 投與群(Cd+Ra, retinol acetate 50,000 I.U./㎏)으로 구분하였다. 시험물질은 시간 간격에 따라 1, 2, 4, 8, 16, 24시간, 2, 4. 7일, 2, 4, 8, 16주에 投與하였으며, 관찰한 결과는 다음과 같다. 1. 카드뮴投與 後 4시간에 血中 카드뮴含量이 0.972∼l.256 ㎍/g으로 對照群의 0.004㎍/g에 비하여 높아졌으며, 投與 2주까지는 肝臟의 카드뮴含量이 23.76∼24.84 ㎍/g으로 腎臟의 20.53∼22.03 ㎍/g보다 높았으나, 8주 후부터는 肝臟의 카드뮴含量(82.48∼86.37 ㎍/g)보다 腎臟의 카드뮴含量(98.0∼109.8 ㎍/g)이 더 높았다. 따라서, 카드뮴은 投與 後 4시간에 혈액 중에 높게 나타나고, 점차 肝臟으로 이동하며 다시 肝臟에서 腎臟으로 이동함으로써, 8주 이후에는 肝臟보다 腎臟의 카드뮴 蓄積量이 많아지는 것으로 나타났다. 2. 카드뮴投與에 의한 血淸중의 酸素 變化에서 ALT(Alanine Aminotransferase), AST(Aspartate Aminotransferase) 活性度는 投與 後 1주일부터 각자 60.3∼73.0 U/ℓ, 135.5∼149.8 U/ℓ로 증가했지만 肝臟 毒性을 일으키는 정도는 아니었으며, glucose는 投與 8주에 對照群(113.8 ㎎/㎗)에 비하여 Cd+Ra群을 제외한 모든 群에서 72.8∼77.5 ㎎/㎗의 낮은 결과를 보여 대조군과 투여군간의 통계학적 有意性(p<0.05)을 보였다. BUN(blood urea nitrogen)은 投與 4주에 對照群의 19.3 ㎎/㎗에 비하여 Cd, Cd+Dds群에서 24.8∼25.8 ㎎/㎗ 有意한 증가를 보였으나, Cd+Ra群에서는 投與 16주에, Cd群의 33.2 ㎎/㎗에 비하여 27.3 ㎎/㎗으로 有意하게(p<0.05) 낮았다. Creatinine은 모든 試驗群에서 投與 8주에 對照群의 0.77 ㎎/㎗에 비하여 0.98∼1.38 ㎎/㎗로 높은 증가를 보였으며(p<0.05), 16주에는 Cd群의 1.20 ㎎/㎗에 비하여 Cd+Ra群은 1.02 ㎎/㎗로 有意하게 낮은 결과를 보였다(p<0.05). 3. 病理組織學적 檢査 결과 Cd群과 Cd+Ra群에서는 投與 8주부터 腎臟細尿管의 內腔에 好酸性顆粒이나 尿圓柱(urinary cast)가 관찰되었으며, Cd+Dds群은 腎臟 近位細尿管 上皮細胞에 顯著한 變性壤死가 나타났다. ??丸은 카드뮴投與 8주부터 精細管(seminiferous tubule) 사이의 間質組織이 好酸性 液體의 貯留에 의해 확장되어 주위의 精細管이 심하게 萎縮되는 등의 소견을 보였다. Cd+Dds群과 Cd+Ra群에서는 Cd群보다 病變이 微弱하였다. 카드뮴을 2주동안 投與한 랫드에서 腎臟의 電子顯微鏡 所見은 近位曲細尿管 細胞의 細胞質 腫脹, 미토콘드리아의 變性, 蛋白質 小球의 증가, 毛細血管 內皮細胞의 細胞質 腫脹과 空砲形成등이 관찰되기 시작하였으며, 投與 8주에 나타난 腎臟細尿管 內腔의 無形質 小球는 탈락된 變性 上皮細胞인 것으로 확인되었다. 4. HSP_(70)은 카드뮴投與 後 2시간부터 發顯이 증가되어, 48시간까지 持續되며, 이후에 原狀態로 恢復되는 것으로 나타났다. 위와같은 HSP_(70)의 發顯은 diallyl disulfide와 retinol acetate 投與에 의해 영향을 받지 않았다. 腎臟에서의 HSP_(70) 發顯은 絲球體와 細尿管上皮細胞에서 주로 發顯되는 것으로 나타났다. 이상의 결과로부터, 랫드에 카드뮴을 投與하였을 때 HSP_(70) 發顯은 投與 後 2∼4시간 내에 나타나는 迅速한 反應임을 알 수 있었고, 마늘유(diallyl disulfide) 投與는 카드뮴에 의한 腎臟損傷을 促進시키나, 비타민 A(retinol acetate) 投與는 카드뮴에 의한 腎臟損傷을 抑制시키는 효과를 보였다. 카드뮴에 의한 ??丸손상은 마늘유(diallyl disulfide) 흑은 비타민 A(retinol acetate)를 投與함으로써 損傷 防禦效果를 보였다.

Heat shock protein (HSP) 70, the highly conserved stress protein families, plays important roles in protecting cells against heat and other stresses in most animal species. In the present study, we identified and characterized four Hsp70 (RuHSP4, RuHSC70, RuHSP12A, RuGRP78) family proteins based on the expressed sequence tag (EST) analysis of the Korean rose bitterling R. uyekii cDNA library. The deduced RuHSP70 family has high amino acid identities of 72-99% with those of other species. Phylogenetic analysis revealed that RuHsp70 family clustered with fish groups (HSP4, HSC70, HSP12A, GRP78) proteins. Quantitative RT-PCR analysis showed the specific expression patterns of RuHsp70 family members in the early developmental stages and several tissues in Korean rose bitterling. The expression of 4 groups of Hsp70 family was detected in all tested tissue. Particularly, Hsp70 family of Korean rose bitterling is highly expressed in hepatopancreas and sexual gonad (testis and ovary). The expression of Hsp70 family was differentially regulated in accordance with early development stage of Rhodeus uyekii.

We used a microarray dataset that is deposited in the public database to evaluate plant responses to heat stress and selected two genes, OsSHSP1 (Os03g16030) and OsSHSP2 (Os01g04380), that are highly expressed under heat stress in rice. OsSHSP1 and OsSHSP2 gene transcripts were highly induced in response to salt and drought. In addition, OsSHSP1 and OsSHSP2 gene transcripts were induced under ABA and SA. Subcellular localization of proteins of 35S::OsSHSP1 were associated with the cytosol, whereas those of and 35S::OsSHSP2 were associated with the cytosol and nucleus. Heterogeneous overexpression of both genes exhibited higher germination rates than those of wild-type plants under the salt treatment, but not under heat or drought stress. The network of both genes harboring 9 sHSPs as well as at least 13 other chaperone genes might support the idea of a role for sHSPs in the chaperone network. Our findings might provide clues to shed light on the molecular functions of OsSHSP1 and OsSHSP2 in response to abiotic stresses, especially heat stress.