• Title/Summary/Keyword: Deletion mutants

Search Result 175, Processing Time 0.029 seconds

Domain Function and Relevant Enzyme Activity of Cycloinulooligosaccharide Fructanotransferase from Paenibacillus polymyxa (Paenibacillus polymyxa Cycloinulooligosaccharide Fructanotransferase의 효소 활성에 미치는 각 Domain의 역할)

  • You Dong-Ju;Park Jung-Ha;You Kyung-Ok;Nam Soo-Wan;Kim Kwang-Hyeon;Kim Byung-Woo;Kwon Hyun-Ju
    • Microbiology and Biotechnology Letters
    • /
    • v.34 no.3
    • /
    • pp.278-287
    • /
    • 2006
  • Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cycloinulooligosaccharides (cyclofructan, CF) of ${\beta}-(2{\to}1)$-linked D-fructofuranose as well as hydrolysis of cyclofructan. Sequences analysis indicated that CFTase was divided into five distinct regions containing three repeated sequences (R1, R3, and R4) at the N-terminus and C-terminus. Each domain function was investigated by comparison of wild type CFTase enzyme (CFT148) and deletion mutant proteins (CFT108: R1 and R3 deletion; CFT130: R4 deletion; and CFT88: R1, R3, and R4 deletion) of CFTase. The CFT108 mutant had both CFTase and CF hydrolyzing activity as CFT148 did. CFTase activities and CF hydrolysing activities were disappeared in CFT130 and CFT88 mutants. These results indicated that the C-terminal R4 region of P. polymyxa CFTase is necessary for cyclization and hydrolyzing activity.

Linker Scanning Analysis of the BPV-1 Upstream Regulatory Region

  • Kim, Hee-Dai;Rho, Jae-Rang;Choe, Joon-Ho
    • BMB Reports
    • /
    • v.28 no.4
    • /
    • pp.368-373
    • /
    • 1995
  • The upstream regulatory region (URR) of bovine papillomavirus type 1 (BPV) contains promoters and a conditional transcriptional enhancer that is trans-activated by the viral E2 protein. After deleting the 5' and 3' ends of BPV URR, BamHI linkers were inserted into several positions of BPV URR without causing an addition or a deletion of URR sequences. Most linker scanning mutations did not show any effects on the transcription of P7940 and P89 promoters in BPV URR. However, several mutants showed reduced transcriptional activities. Based on our results we found that the AP-2 and Sp1 binding sites were important for basal level transcription of BPV URR in the absence of the E2 protein and that the CTF/NF-1 site is dispensable for E2 transactivation of BPV URR transcription.

  • PDF

Quorum Sensing of Rhodobacter sphaeroides Negatively Regulates Cellular Poly-$\beta$-Hydroxybutyrate Content Under Aerobic Growth Conditions

  • Lee, Jeong-K.;Kho, Dhong-Hyo;Jang, Ji-Hee;Kim, Hye-Sun;Kim, Kun-Soo
    • Journal of Microbiology and Biotechnology
    • /
    • v.13 no.3
    • /
    • pp.477-481
    • /
    • 2003
  • The community escape response of Rhodobacter sphaeroides is exerted through the action of CerR and CerI, which code for a LuxR-type regulatory protein and acylhomoserine lactone synthase, respectively. Deletion of chromosomal DNA including cerR and cerI (mutant RI) or insertional interruption of cert (mutant AP3) resulted in two-fold increase in the cellular poly-${\beta}$-hydroxybutyrate (PHB) content In comparison with the wild-type under aerobic growth conditions. The PHB synthase (PhbC) activities of the cer mutants were doubled, and the enzyme expression was regulated at the level of phbC transcription. Thus, CerR, possibly in response to autoinducer (AI), appears to modulate the PHB content of aerobically grown cells by downregulating phbC transcription.

Mutational Analysis of Cucumber Mosaic Virus Movement Protein Gene

  • You, Jin-Sam;Baik, Hyung-Suk;Paek, Kyung-Hee
    • BMB Reports
    • /
    • v.32 no.1
    • /
    • pp.82-85
    • /
    • 1999
  • The movement protein of cucumber mosaic virus (CMV) is required for cell-to-cell movement of viral RNA. The movement of viral RNA occurs through the plant intercellular connection, the plasmodesmata. The viral movement protein was known to be multi-functional. In this work, a series of deletion mutants of CMV movement protein gene were created to identify the functional domains. The mutated movement proteins were produced as inclusion body in E. coli, and purified and renatured. A polyclonal antibody was raised against the CMV-Kor strain (Korean isolate) movement protein expressed in E. coli. The ability of the truncated proteins to bind to ssRNA was assayed by UV cross-linking and gel retardation analyses. The results indicate that the domain between amino acids 118 and 160 of CMV movement protein is essential for ssRNA binding.

  • PDF

Mapping of the equine herpesvirus type 1 immediate-early protein interaction domain within the general transcription factor human TFIIB

  • Jang, Hyung-Kwan;Cho, Jeong-Gon;Song, Hee-Jong
    • Korean Journal of Veterinary Service
    • /
    • v.25 no.4
    • /
    • pp.333-346
    • /
    • 2002
  • We previously reported that the equine herpesvirus type 1(EHV-1) immediate-early protein(IE protein) physically interacts with the general transcription factor human TFIIB(Jang et al, J Virol 75:10219-10230, 2001). The interaction between the IE protein and TFIIB is necessary for the IE protein to efficiently transactivate the early TK and late IR5 EHV-1 promoters. A panel of deletion and truncation mutants of the TFIIB gene was constructed and employed in protein-binding assays to map the IE protein-binding domain within TFIIB. Evidence is presented that the first direct repeat of TFIIB interacts specifically with the EHV-1 IE protein.

A Rice Blast Fungus Alpha-N-Arabinofuranosidase B Elicits Host Defense in Rice

  • Kim, Sun-Tae
    • 한국균학회소식:학술대회논문집
    • /
    • 2015.11a
    • /
    • pp.23-23
    • /
    • 2015
  • Rice blast disease caused by M. oryzae is the most devastating fungal disease in rice. During the infection process, M. oryzae secretes a large number of glycosyl hydrolase (GH) proteins into the apoplast to digest host cell wall and assist fungal ingress into host tissues. In this study, we identified a novel M. oryze arabinofuranosidase B (MoAbfB) which is secreted during fungal infection. Live-cell imaging exhibited that fluorescent labeled MoAbfB was highly accumulated in fungal invasive structures such as appressorium, tips of penetration peg, biotrophic interfacial complex (BIC), as well as invasive hyphal tip. Deletion of MoAbfB mutants extended biotrophic phase followed by enhanced disease severity, whereas, over-expression of OsMoAbfB mutant induced rapid defense responses and enhanced rice resistance to M. oryzae infection. Furthermore, exogenous treatment of MoAbfB protein showed inhibition of fungal infection via priming of defense gene expression. We later found that the extract of MoAbfB degraded rice cell wall fragments could also induce host defense activation, suggesting that not MoAbfB itself but oligosaccharides (OGs) derived from MoAbfB dissolved rice cell wall elicited rice innate immunity.

  • PDF

Mesenchymal Smad4 mediated signaling is essential for palate development (구개 형성과정에서 간엽 내 Smad4 매개 신호전달의 역할)

  • Yoon, Chi-Young;Baek, Jin-A;Cho, Eui-Sic;Ko, Seung-O
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
    • /
    • v.36 no.6
    • /
    • pp.460-465
    • /
    • 2010
  • Introduction: A cleft palate is a common birth defect in humans with an incidence of 1/500 to 1/1,000 births. It appears to be caused by multiple genetic and environmental factors during palatogenesis. Many molecules are involved in palate formation but the biological mechanisms underlying the normal palate formation and cleft palate are unclear. Accumulating evidence suggests that transforming growth factor $\beta$/bone morphogenetic proteins (TGF-$\beta$/BMP) family members mediate the epithelial-mesenchymal interactions during palate formation. However, their roles in palatal morphogenesis are not completely understood. Materials and Methods: To understand the roles of TGF-$\beta$/BMP signaling in vivo during palatogenesis, mice with a palatal mesenchyme- specific deletion of Smad4, a key intracellular mediator of TGF-$\beta$/BMP signaling, were generated and analyzed using the Osr2Ires-Cre mice. Results: The mutant mice were alive at the time of birth with open eyelids and complete cleft palate but died within 24 hours after birth. In skeletal preparation, the horizontal processes of the palatine bones in mutants were not formed and resulted in a complete cleft palate. At E13.5, the palatal shelves of the mutants were growing as normally as those of theirwild type littermates. However, the palatal shelves of the mutants were not elevated at E14.5 in contrast to the elevated palatal shelves of the wild type mice. At E15.5, the palatal shelves of the mutants were elevated over the tongue but did not come in contact with each other, resulting in a cleft palate. Conclusion: These results suggest that mesenchymal Smad4 mediated signaling is essential for the growth of palatal processes and suggests that TGF-$\beta$/BMP family members are essential regulators during palate development.

Biological Function of Single Chain Glycoprotein Hormone Mutants

  • Min, Kwan-Sik;Chang, Yoo-Min;Chang, Sun-Hwa;Lee, Hyen-Gi;Lee, Yun-Gun;Chang, Won-Kyong;Cheong, Il-Cheong
    • Proceedings of the KSAR Conference
    • /
    • 2001.03a
    • /
    • pp.54-54
    • /
    • 2001
  • Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family which includes FSH, hCG, TSH. These hormone family is characterized by a heterodimeric structure composed a common $\alpha$-subunit noncovalently linked to a hormone specific $\beta$-subunit. The correct conformation of the heterodimer is also important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduciton. To determine $\alpha$ and $\beta$-subunits can be synthesized as a single polypeptide chain (tethered-hCG) and also display biological activity, the tethered-hCG molecule by fusing the carboxyl terminus of the hCG $\beta$-subunit to the amino terminus of the $\alpha$-subunit was constructed and transfected into chinese hamster ovary (CHO-K1) cells. We also constructed C-terminal deletion mutants (D9l, D89, D88, D87, D86, D84, D83) of single chain hCG to determine the biological function (secretion, LH-activity, receptor binding, cAMP production) of these mutants. Between six and eight stably transfected pools of cells expressing wild type and mutant hCGs were selected for neomycin resistant. The hCGs secreted by the stably transfected cells into serum-free media were collected and quantified by radioimmunoassay, as described in protocol (DPC(hCG IRMA). LH activity was in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells. The tethered-wthCG was efficiently secreted and showed similar LH-like activity to the dimeric hCG. The D83hCG mutant was not detected in this assay. It is suggest that hCG C-terminal part is very important for hCG secretion. Now, we checking the LH-like activity of these mutant hCGs. These data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

  • PDF