• BMB Reports
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• 제46권1호
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• pp.53-58
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• 2013

We constructed deletion mutants and seven point mutants by polymerase chain reaction to investigate the specificity of feline foamy virus integrase functional domains. Complementation reactions were performed for three enzymatic activities such as 3'-end processing, strand transfer, and disintegration. The complementation reactions with deletion mutants showed several activities for 3'-end processing and strand transfer. The conserved central domain and the combination of the N-terminal or C-terminal domains increased disintegration activity significantly. In the complementation reactions between deletion and point mutants, the combination between D107V and deletion mutants revealed 3'-end processing activities, but the combination with others did not have any activity, including strand transfer activities. Disintegration activity increased evenly, except the combination with glutamic acid 200. These results suggest that an intact central domain mediates enzymatic activities but fails to show these activities in the absence of the N-terminal or C-terminal domains.

• BMB Reports
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• 제36권4호
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• pp.344-348
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• 2003

Protein kinase CKII is composed of two catalytic ($\alpha$ or $\alpha$') subunits and two regulatory ($\beta$) subunits. The $CKII{\beta}$ subunit is thought to mediate the tetramer formation and interact with other target proteins. However, its physiological function remains obscure. In this study, point mutants of $CKII{\beta}$ that are defective for the L41 binding were isolated by using the reverse two-hybrid system. A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of $CKII{\beta}$ are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for $CKII{\beta}$ homodimerization. Two point mutants, R75 and R83, of $CKII{\beta}$ interacted with L5, topoisomerase $II{\beta}$, and CKBBP1/SAG, but not with the wild-type $CKII{\beta}$. This indicates that $CKII{\beta}$ homodimerization is not a prerequisite for its binding to target proteins. These $CKII{\beta}$ point mutants may be useful in exploring the biochemical physiological functions of $CKII{\beta}$.

• 약학회지
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• 제49권3호
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• pp.198-204
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• 2005

Human foamy virus (HFV) integrase mediates integration of viral c-DNA into cellular DNA. In this process, HFV integrase recognizes its own viral DNA specifically and catalyzes insertion of viral c-DNA. In order to study catalytic domains and residues, three deletion mutants and two point mutants of HFV integrase were constructed and analyzed with respect to enzymatic activities. The C-terminal deletion mutant showed decreased enzymatic activities while the N-terminal deletion mutant lost the activities completely, indicating that the N-terminal domain is more important than the C-terminal domain in enzymatic reaction. The point mutants, in which an aspartic acid at the 164th position or a glutamic acid at the 200th position of the HFV integrase protein was changed to an alanine, lost the enzymatic activities completely. However, they were well complemented with other defective deletion mutants to recover enzymatic activities partially. Therefore, these results suggest that the aspartic acid and glutamic acid at the respective 164th and 200th positions are catalytic residues for enzymatic reaction.

• 미생물학회지
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• 제20권4호
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• pp.173-182
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• 1982

From FGSC 159 strain of Aspergillus nidulans, temperature sensitive mutants that are defective in growth and differentiation have been isolated by N-methyl-N'-nitroN-nitrosoguanidine (NTG) treatment. The optimum concentration of NTG and incubation time to get the highest mutation frequency was $100{\mu}g$ per ml and 1 hour, respectively. The survival frequency was 1%. Among the isolated mutants, five strains that were affected in early steps of differentiation were selected for further studies and named smK, smY, smB, smF, and smZ. The execution point of each mutant was determined and the growing pattern of each mutant at the restrictive temperature was observed under the microscope. Growth of mutant was arrested near at the execution point. From genetic analysis, each temperature-sensitive mutants was thought to have a single recessive gene. The genes of smK, smY, smB, smF, and smZ are linked to the chromosome VII, IV, VIII, I, and VI, respectively. It can be concluded that the genes controlling the differentiation are widely dispersed in the genome. From the results of mutant, smK, it is considered that a single gene can affect a function (functions) which act(s) at two different steps during differentiation.

• Molecules and Cells
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• 제19권1호
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• pp.124-130
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• 2005

Protein kinase CKII is composed of two catalytic (${\alpha}$ or ${\alpha}^{\prime}$) subunits and two regulatory (${\beta}$) subunits. The ${\beta}$ subunit mediates tetramer formation through ${\beta}-{\beta}$ homodimerization and ${\alpha}-{\beta}$ heterodimerization. In a previous study R26 and R75, point mutants of $CKII{\beta}$ defective in ${\beta}-{\beta}$ dimerization, were isolated. In the present work we characterized these $CKII{\beta}$ mutants in vitro. Purified R26 and R75 bound to $CKII{\alpha}$ but were defective in binding to $CKII{\beta}$. R75 stimulated the catalytic activity of CKII whereas R26 gave little stimulation, and poly-L-lysine increased the stimulation of catalytic activity by R26 or R75. Circular dichroism and intrinsic fluorescence data pointed to different conformational changes in R26 and R75. Molecular modeling of these mutants provides an explanation of the difference in their ability to interact with $CKII{\beta}$ and to activate $CKII{\alpha}$.

• Molecules and Cells
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• 제19권2호
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• pp.246-255
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• 2005

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous N-terminal and C-terminal domains.

• 미생물학회지
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• 제37권1호
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• pp.28-36
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• 2001

본 연구에서는 세포주기의 Gl/S기에 관련된 유전자의 작용기작을 이해하기 위하여 출아효모인 Saccharomyces cerevisiae를 사용하여 세포주기 진행 및 조절에 관련된 변이주를 분 리하여 특성화하는 연구를 수행하였다. 먼저 새로운 변이주를 분리하기 위해서 HeLa 세포와 출아효모에서 Gl을 유발하는 CPO(ciclopirox olamine) 약제에 대해 감수성을 보이는 변이주를 선별하였다. 그 결과, 총 31개의 CPO에 대한 감수성 변이주들이 분리되었고, ciclopirox olamine sensitivity의 첫 글자를 따서 con mutants라 명명하였다. cos 변이주들의 표현형의 특성을 결정하기 위해 MMS(methylmethane sulfonate)와 HU(hydrsxyurea)에 대한 감수성을 조사하여 그 성질에 따라 4개의 group으로 나누었다. Group I에는 cos27, cos28, cos32, cos33, cos36, cos37, cos40, cos42, cos46, cos50, cos52, cos53 변이주가 포함되고, 이들은 MMS와 HU 두 약제 모두에 대해서 감수성을 보여 S기 checkpoint에 관련된 것으로 보이고, Group II는 cos43, cos48 변이주로 MMS에 대해서 감수성을 지녀 G1기 또는 G2기 checkpoint에 관련된 것으로 추측된다. Group III 변이주에는 cos35, cos47, cos54, cos55, cos56이 포함되고 HU에 대해서 감수성을 보여 S기에 관련된다고 보여지며, Group IV 변이 주는 cos29, cos30, cos31, cos34, cos38, cos39, cos41, cos44, cos45, cos49, cos51, cos57로서 단지 CPO에 대해서만 감수성을 나타냈다. 더욱이 변이주의 최종표현형을 형광현미경을 이용하여 조사하여, S기 checkpoint에 관련된 것으로 보이는 cos37 변이주를 확인하였다. 또한, 변이주와 야생주의 이형접합체인 이배체를 만들어 변이주의 유전학적 분석을 수행하였다.

• 대한전자공학회:학술대회논문집
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• 대한전자공학회 2009년도 정보 및 제어 심포지움 논문집
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• pp.135-137
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• 2009

Mathematical models for describing the Human Immunodeficiency Virus(HIV) infection can be devised to better understand how the HIV causes Acquired Immune Deficiency Syndrome(AIDS). The HIV models can then be used to find clues to curing AIDS from a control theoretical point of view. Some models take Cytotoxic T Lymphocytes(CTL) response to HIV infection into account, and others consider mutants against the drugs. However, to the best of our knowledge, there has been no model developed, which describes CTL response and mutant HIV together. Hence we propose a unified model to consider both of these. On the basis of the resulting model, we also present a Model Predictive Control(MPC) scheme to find an optimal treatment strategy. The optimization is performed under the assumption that the Structured Treatment Interruption(STI) policy is employed.

• Molecules and Cells
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• 제37권2호
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• pp.140-148
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• 2014

We identified four basic amino acid residues as nuclear localization signals (NLS) in the C-terminal domain of the prototype foamy viral (PFV) integrase (IN) protein that were essential for viral replication. We constructed seven point mutants in the C-terminal domain by changing the lysine and arginine at residues 305, 308, 313, 315, 318, 324, and 329 to threonine or proline, respectively, to identify residues conferring NLS activity. Our results showed that mutation of these residues had no effect on expression assembly, release of viral particles, or in vitro recombinant IN enzymatic activity. However, mutations at residues 305 (R ${\rightarrow}$ T), 313(R ${\rightarrow}$ T), 315(R ${\rightarrow}$ P), and 329(R ${\rightarrow}$ T) lead to the production of defective viral particles with loss of infectivity, whereas non-defective mutations at residues 308(R ${\rightarrow}$ T), 318(K ${\rightarrow}$ T), and 324(K ${\rightarrow}$ T) did not show any adverse effects on subsequent production or release of viral particles. Sub-cellular fractionation and immunostaining for viral protein PFV-IN and PFV-Gag localization revealed predominant cytoplasmic localization of PFV-IN in defective mutants, whereas cytoplasmic and nuclear localization of PFV-IN was observed in wild type and non-defective mutants. However sub-cellular localization of PFV-Gag resulted in predominant nuclear localization and less presence in the cytoplasm of the wild type and non-defective mutants. But defective mutants showed only nuclear localization of Gag. Therefore, we postulate that four basic arginine residues at 305, 313, 315 and 329 confer the karyoplilic properties of PFV-IN and are essential for successful viral integration and replication.

• BMB Reports
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• 제43권3호
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• pp.205-211
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• 2010

Sprouty (Spry) proteins have previously been suggested as negative regulators of the MAPK pathway through interaction with Raf-1. However, the molecular basis of this inhibition has not been elucidated. In this study, we used cells expressing FLAGtagged Raf-1 with point mutations at known phosphorylation sites to reveal that activation of Raf-1 mutants does not correlate with their degree of interaction with Spry2. The association of Raf-1 with Spry2 in intact cells was further corroborated by immunofluorescence colocalization. Additionally, there was no significant change observed in the strength of interaction between Raf-1 mutants and Spry2 after paclitaxel treatment despite differences in the activation levels of these mutants. Thus, our study provides the evidence that Spry2 does not directly regulate Raf-1 kinase activity, but instead acts as a scaffolding protein that assists interactions between Raf-1 kinase and its direct regulators.