• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.41-41
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• 2001

Chromatin configuration and microtubule assembly were determined in porcine maturing and activated oocytes following intracytoplasmic sperm injection. Microtubule localization was confirmed using a mouse monoclonal antibody to $\alpha$-tubulin and detected using a fluorescent labeled goat anti-mouse secondary antibody. DNA was stained with propidium iodide. The image of microtubules and chromatin was captured using laser scanning confocal microscope. In germinal vesicle stage oocyte, sperm chromatin remained condensation and sperm derived microtubules were not observed at 8 to 12 h after sperm injection. At 24 h after injection, the sperm nucleus developed to the metaphase chromatin along the metaphase structure of female nucleus. In some metaphase I stage oocytes, sperm chromatin decondensed at 8 h to 12 h after injection, sperm aster was seen soon after sperm injection. At 24 h after sperm injection into metaphase I stage oocyte, male chromatin developed to the metaphase chromatin while female chromatin extruded first polar body and formed the metaphase chromatin. At 12 to 15 h after sperm injection into preactivated oocytes, condensed sperm nucleus was located in close proximity of female pronucleus. However, the condensed nucleus did not fuse with female pronucleus. In preactivated ocytes, injected sperm remained condensation, a few sperm organized small microtubular aster. Instead, maternal derived microtubules were organized near the female chromatin, which seem to move condensed male chromatin near to the female pronucleus. These results suggest that sperm nuclear decondensing activity and nucleation activity of centrosome during fertilization are cell cycle dependent. In absence of male functional centrosome, female origin centrosome takes over the role of microtubule nucleation for nuclear movement.

• BMB Reports
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• 제42권4호
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• pp.227-231
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• 2009

The eukaryotic genome forms a chromatin structure that contains repeating nucleosome structures. Nucleosome packaging is regulated by chromatin remodeling factors such as histone chaperones. The Saccharomyces cerevisiae H3/H4 histone chaperones, CAF-1 and Asf1, regulate DNA replication and chromatin assembly. CAF-1 function is largely restricted to non-transcriptional processes in heterochromatin, whereas Asf1 regulates transcription together with another H3/H4 chaperone, HIR. This study examined the role of the yeast H3/H4 histone chaperones, Asf1, HIR, and CAF-1 in chromatin dynamics during transcription. Unexpectedly, CAF-1 was recruited to the actively transcribed region in a similar way to HIR and Asf1. In addition, the three histone chaperones genetically interacted with Set2-dependent H3 K36 methylation. Similar to histone chaperones, Set2 was required for tolerance to excess histone H3 but not to excess H2A, suggesting that CAF-1, Asf1, HIR, and Set2 function in a related pathway and target chromatin during transcription.

• Mycobiology
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• 제41권1호
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• pp.1-12
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• 2013

Msi1-like (MSIL) proteins, which are eukaryote-specific and contain a series of WD40 repeats, have pleiotropic roles in chromatin assembly, DNA damage repair, and regulation of nutrient/stress-sensing signaling pathways. In the fungal kingdom, the functions of MSIL proteins have been studied most intensively in the budding yeast model Saccharomyces cerevisiae, an ascomycete. Yet their functions are largely unknown in other fungi. Recently, an MSIL protein, Msl1, was discovered and functionally characterized in the pathogenic yeast Cryptococcus neoformans, a basidiomycete. Interestingly, MSIL proteins appear to have redundant and unique roles in both fungi, suggesting that MSIL proteins may have evolutionarily divergent roles in different parts of the fungal kingdom. In this review, we will describe the current findings regarding the role of MSIL proteins in fungi and discuss future directions for research on this topic.

• BMB Reports
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• 제48권12호
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• pp.685-690
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• 2015

The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modifications. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases.

• Animal cells and systems
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• 제13권2호
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• pp.235-245
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• 2009

Embryonic stem (ES) cells have a capability to generate all types of cells. However, the mechanism by which ES cells differentiate into specific cell is still unclear. Using microarray technology, the differentiation process in mouse embryonic stem cells was characterized by temporal gene expression changes of mouse ES cells during differentiation in a monolayer culture. A large number of genes were differentially regulated from 1 day to 14 days, and less number of genes were differentially expressed from 14 days to 28 days. The number of up-regulated genes was linearly increased throughout the 28 days of in vitro differentiation, while the number of down-regulated genes reached the plateau from 14 days to 28 days. Most differentially expressed genes were functionally classified into transcriptional regulation, development, extra cellular matrix (ECM),cytoskeleton organization, cytokines, receptors, RNA processing, DNA replication, chromatin assembly, proliferation and apoptosis related genes. While genes encoding ECM proteins were up-regulated, most of the genes related to proliferation, chromatin assembly, DNA replication, RNA processing, and cytoskeleton organization were down-regulated at 14 days. Genes known to be associated with embryo development or transcriptional regulation were differentially expressed mostly after 14 days of differentiation. These results indicate that the altered expression of ECM genes constitute an early event during the spontaneous differentiation, followed by the inhibition of proliferation and lineage specification. Our study might identify useful time-points for applying selective treatments for directed differentiation of mouse ES cells.

• 한국가축번식학회지
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• 제24권2호
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• pp.143-154
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• 2000

본 연구는 생식보조기법을 시행한 불임환자로부터 얻은 난자를 일반적인 수정법과 세포질내 정자직접주입법으로 수정을 유도한 다음 정상수정에 실패한 난자에 대한 미세소관과 염색체의 형태학적 차이를 laser scanning confocal microscope를 이용하여 비교분석하고자 실시하였다. 일반적 수정법 혹은 세포질내 정자직접주입법 실시 후 18시간째에 해부현미경 하에서 난자를 관찰하였을 때 전핵형성에 실패한 미수정란, 한 개의 전핵 또는 3개이상의 전핵의 형성이 관찰된 이상수정란으로 구분하여 연구를 실시하였다. 미세소관의 관찰을 위해서 (-tubulin antibody를 반응시킨 후 형광물질이 부착된 2차항체와 반응시킨 후 관찰하였으며 염색체의 관찰을 위해서는 propidium iodide로 염색한 다음 confocal microscope 하에서 관찰하였다. 연구결과 대부분의 난자는 수정과정중에 있었으나 일부의 난자에서는 특정단계에서 정지되어 있는 것이 관찰되었다. 즉, 감수분열 중기에서 정자의 침입이 이루어지지 않은 경우, 정자의 침입은 이루어졌으나 sperm aster 형성이 불완전한 경우, 웅성 및 자성전핵의 형성에 실패한 경우 및 전핵의 위치가 불완전한 경우 등이 관찰되었고 이들 난자의 경우 높은 비율로 미세소관과 염색체의 이상이 관찰되었다. 이상의 연구결과로 미루어 볼 때 생식보조기법의 시술과정에서 채취되는 난자의 수정실패의 원인은 세포골격기관 특히 미세소관의 이상과 염색체의 이상에 기인되는 것으로 사료되면 이러한 세포골격 구성물질의 이상에 대해서는 추후에 세포조직학적 또는 분자생물학적 분석이 필요하다고 하겠다.

• BMB Reports
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• 제31권5호
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• pp.484-491
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• 1998

Acetylation of lysine residues within the aminoterminal domains of the core histones plays a critical role in chromatin assemhly as well as in regulation of gene expression. To study the biochemical function of histone acetylation, we have cloned a cDNA encoding the catalytic subunit of human histone acetyltransferase, Hat1. Analysis of the predicted amino acid sequence of human Hat1 revealed an open reading frame of 419 amino acids with a calculated molecular mass of 49.5 kDa and an isoelectric point of 5.5. The amino acid sequence of human Hat1 is homologous to those of known and putative Hat1 proteins from various species throughout the entire open reading frame. The recombinant human Hat1 protein expressed in bacteria possesses histone H4 acetyltransferase activity in vitro. Both RbAp46 and RbAp48, which participate in various processes of histone metabolism, enhance the histone acetyltransferase activity of the recombinant human Hat1, indicating that they are both able to functionally interact with the human Hat1 in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제24권3호
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• pp.301-309
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• 1997

본 연구에서는 체외수정, 난자내 정자 직접주입, 난자내 정자 두부 미두 주입 후의 핵과 미세소관의 변화를 관찰하였다. 핵과 미세소관의 움직임은 형광염색을 실시한 후 공초점주사현미경을 이용하여 관찰하였다. 체외수정에서 관찰된 바와 동일하게 정자를 난자에 직접주입 한 직후 정자 중편부에서 성상체가 형성되었고, 이 성상체에 의해 자성 웅성 전핵이 융합되는 것으로 관찰되었다. 그러나 난자내 정자를 직접주입하였을 경우 웅성전핵으로 발달하는 비율이 낮았다. 이는 주입된 정자가 원형질막과 perinuclear theca에 싸인 체 난자내로 들어가 난자내의 sperm nucleus decondensing factor와 정자 핵과의 반응이 억제되기 때문으로 생각된다. 정자 두부 만을 주입하였을 경우 성상체가 형성되지 않았지만 자성 웅성 전핵 사이 또는 그 주위에서 두터운 미세소관층이 관찰이 되었다. 따라서 소에 있어서는 정자의 중편부에 위치하여 microtubule organizing center (MTOC)의 역할을 하는 중심립 또는 중심체 없이도 모계에서 유래된 미세소관이 형성되어 이것이 전핵의 융합과 세포분열에 관여하는 것으로 생각된다. 정자의 미부 만을 주입하였을 경우 성상체가 형성이 되지 않았으며, 자성핵 사이에 형성된 미세소관과 떨어져서 관찰되었다. 따라서 주입된 정자의 꼬리는 미세소관형성과 관련이 없는 것으로 생각된다. 이러한 결과는 소에 있어서, 수정 시 정자로부터 유래되는 중심립 또는 중심체가 없이도 미세소관을 형성하여 미세소관에 의해 이후의 배발달이 정상적으로 일어남을 보여주고 있다.

• Molecules and Cells
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• 제27권1호
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• pp.1-3
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• 2009

Mitotic spindle mediates the segregation of chromosomes in the cell cycle and the proper function of the spindle is crucial to the high fidelity of chromosome segregation and to the stability of the genome. Nucleation of microtubules (MTs) from centrosomes and chromatin represents two well-characterized pathways essential for the assembly of a dynamic spindle in mitosis. Recently, we identified a third MT nucleation pathway, in which existing MTs in the spindle act as a template to promote the nucleation and polymerization of MTs, thereby efficiently amplifying MTs in the spindle. We will review here our current understanding on the molecular mechanism, the physiological function and the cell-cycle regulation of MT amplification.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.51-51
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• 2002

Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we compared chromatin, r-tubulin and microtubule organization in porcine oocytes following somatic cell nuclear transfer and parthenogenetically activation in order to clarify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. (omitted)