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Yeonhee Kim

Publications and source records attributed to Yeonhee Kim.

6 recordsLinked to original sources
PubMed2005

A versatile and general splitting technology for generating targeted YAC subclones.

Yeast artificial chromosomes (YAC) splitting technology was developed as a means to subclone any desired region of eukaryotic chromosomes from one YAC into new YACs. In the present study, the conventional YAC splitting technology was improved by incorporating PCR-mediated chromosome splitting technique and by adding autonomously replicating sequence (ARS) to the system. To demonstrate the performance of the improved method, a 60-kb region from within a 590-kb YAC (clone CIC9e2 from Arabidopsis thaliana chromosome 5) that could not be subcloned using the original method was split to convert into a replicating YAC. Two template plasmids, pSK-KCA and pSKCLY, were used to generate two splitting fragments by PCR. Two splitting fragments consisted of telomeric (C(4)A(2))(6) repeats, 400-bp target region, CEN4, H4ARS and Km(r) (selective marker for plant transformants), or CgLEU2. These splitting fragments were introduced into Saccharomyces cerevisiae harboring the 100-kb split YAC generated by splitting of the 590-kb YAC and containing the 60-kb region. Among 12 Leu(+) transformants, four exhibited the expected karyotype in which two newly split 40- and 60-kb chromosomes were generated. These results demonstrate that the improved method can convert a targeted region of a eukaryotic chromosome within a YAC into a replicating YAC.

Arabidopsis↗
PubMed2005

A yeast artificial chromosome-splitting vector designed for precise manipulation of specific plant chromosome region.

A yeast artificial chromosome (YAC) splitting vector, pKI01, was constructed for manipulating plant chromosome fragments cloned as YACs in order to transfer specific regions of the fragments into plant cells. Vector pKI01 consists of Km(r) and ADE2 genes (selective markers for plant and yeast transformants, respectively), inverted telomeric repeats Tr and CEN4. To demonstrate the utility of pKI01, YAC CIC9e2 harboring a 590-kb fragment from Arabidopsis thaliana chromosome 5 was split into specific fragments. A 1-kb target region positioned 100 kb from the right end of the 590 kb fragment was cloned into pKI01. The resultant plasmid, pKY03, was introduced into Saccharomyces cerevisiae harboring YAC CIC9e2. The Ade+ transformants were found to contain two new YACs of 490 and 100 kb, and to lack the original 590 kb YAC, consistent with the expected splitting event. To release the desired middle region of YAC CIC9e2, two additional splitting vectors were constructed, pKY11 and pKY14. By conducting two rounds of splitting, i.e., the first round 100 kb from the right end of YAC CIC9e2 with pKY11 to generate 490 and 100 kb YACs and a second round 50 kb from the right end of the new 490 kb YAC to generate 440 and 50 kb YACs, the middle 50 kb region of a plant chromosome fragment harboring Km(r) was successfully released as a split YAC. These results indicate that YAC splitting vectors as constructed in this study are useful for generating any desired plant chromosome fragment as a YAC for eventual re-introduction into plant cells.

Arabidopsis↗
PubMed2004

Syndecan-4 regulates localization, activity and stability of protein kinase C-alpha.

During cell-matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibres. We have shown previously that the syndecan-4 cytoplasmic domain directly binds to and activates PKC-alpha (protein kinase C-alpha) in vitro [Oh, Woods and Couchman (1997) J. Biol. Chem. 272, 8133-8136]. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, we showed that syndecan-4 interacted with PKC-alpha in vivo and that this interaction was mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increased the extent of interaction between syndecan-4 and PKC-alpha. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increased the level of endogenous PKC-alpha and enhanced the translocation of PKC-alpha into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibited a slowed down-regulation of PKC-alpha in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-alpha immunocomplex kinase assays also showed that syndecan-4 overexpression increased the activity of membrane PKC-alpha. Taken together, these results suggest that syndecan-4 interacts with PKC-alpha in vivo and regulates its localization, activity and stability.

Animals↗
PubMed2003

Decreased syndecan-2 expression correlates with trichostatin-A induced-morphological changes and reduced tumorigenic activity in colon carcinoma cells.

The inhibition of histone deacetylase activity is known to induce morphological changes of transformed cells. In this study, we investigated the effect of the specific HDAC inhibitor, trichostatin A (TSA), on colon carcinoma cell lines. Treatment of human colorectal carcinoma cells, KM1214 and KM12SM, with TSA induced distinct morphological changes. Both cell lines, which normally piled up in layers without clear boundary, became more flattened, and formed monolayers with evident boundaries between cells, with concomitant increased actin filament organization. Cell-cell interaction was not affected much, based on expression level, membrane localization, and interaction of E-cadherin with beta-catenin. In contrast, syndecan-2 expression was dramatically reduced and it was correlated with the morphological changes of colon carcinoma cells. Consistently, downregulation of syndecan-2 expression by antisense cDNA clearly mimicked the morphological changes in KM12SM and reduced anchorage-independent growth of colon cancer cells. All these results indicate that reduced syndecan-2 expression correlates with TSA-induced morphological changes and reduced tumorigenic activity in colon carcinoma cells.

Cadherins↗
PubMed2002

Syndecan-2 mediates adhesion and proliferation of colon carcinoma cells.

Syndecan-2 is a transmembrane heparan sulfate proteoglycan whose function at the cell surface is unclear. In this study, we examined the function of syndecan-2 in colon cancer cell lines. In several colon cancer cell lines, syndecan-2 was highly expressed compared with normal cell lines. In contrast, syndecan-1 and -4 were decreased. Cell biological studies using the extracellular domain of recombinant syndecan-2 (2E) or spreading assay with syndecan-2 antibody-coated plates showed that syndecan-2 mediated adhesion and cytoskeletal organization of colon cancer cells. This interaction was critical for the proliferation of colon carcinoma cells. Blocking with 2E or antisense syndecan-2 cDNA induced G(0)/G(1) cell cycle arrest with concomitantly increased expression of p21, p27, and p53. Furthermore, blocking of syndecan-2 through antisense syndecan-2 cDNA significantly reduced tumorigenic activity in colon carcinoma cells. Therefore, increased syndecan-2 expression appears to be a critical for colon carcinoma cell behavior, and syndecan-2 regulates tumorigenic activity through regulation of adhesion and proliferation in colon carcinoma cells.

Adenocarcinoma↗