Biomedical subjects

J Yim

Publications and source records attributed to J Yim.

At least 19 recordsLinked to original sources

PubMed2001

Expression of the seqA gene is negatively modulated by the HU protein in Escherichia coli.

The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, effectively blocking methylation and therefore preventing rapid re-initiation. The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase. In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type. These results suggest that the HU protein functions as a negative modulator of seqA expression.

Bacterial Outer Membrane Proteins↗

PubMed2000

Molecular characterization of Drosophila melanogaster myo-inositol-1-phosphate synthase.

We have isolated and characterized a cDNA encoding Drosophila melanogaster myo-inositol-1-phosphate synthase (INOS). The deduced Drosophila INOS protein is 50% identical to the Saccharomyces cerevisiae INO1 gene. The putative active site residues are well conserved in Drosophila INOS protein. Southern blot analysis shows that Drosophila INOS gene is a single copy gene. Northern blot analysis reveals that Drosophila INOS gene expresses a 2.0-kb transcript that is more abundant in the head than the body, suggesting that it may be involved in brain function. The recombinant Drosophila INOS protein was expressed in Escherichia coli and the purified protein has proved to have a myo-inositol-1-phosphate synthase activity.

Amino Acid Sequence↗

PubMed2000

5'-Flanking sequence and promoter activity of the rabbit neuronal nitric oxide synthase (nNOS) gene.

We have isolated a rabbit neuronal nitric oxide synthase (nNOS) cDNA encoding a protein of 1,435 amino acids. Using the cDNA clones as probes, the 5'-flanking region of the nNOS gene was isolated from a rabbit genomic DNA library. 5'RACE and primer extension analysis of rabbit brain total RNA mapped multiple transcription initiation sites localized 474-487 bp upstream from the translation start codon. Analysis of 5,197 bp of the 5'-flanking sequence revealed that the rabbit nNOS gene promoter lacks canonical TATA or CCAAT boxes and, instead, contains a GC-rich region and multiple Sp1 sites. Farther from the +1start, various putative cis-elements including AP-1, AP-4, NF-kappaB, STAT, CREB, C/EBP and c-Myc were observed. The functional promoter activity of the 5'-flanking region was demonstrated by its ability to drive the expression of a beta-galactosidase reporter gene in several cell types. Serial deletion analysis of the promoter region revealed that the -291 to -172 region, which contains two Sp1 sites, is essential for basal transcriptional activity. These results suggest that the rabbit nNOS promoter contains characteristics of inducible genes.

5' Untranslated Regions↗

PubMed2000

Epithelial cell-specific laminin 5 is required for survival of early thymocytes.

The gene LamC2 encoding the gamma2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the alpha3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44-subsets of the CD4-CD8- double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin alpha3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.

Animals↗

PubMed2000

Molecular characterization of Drosophila melanogaster dihydropteridine reductase.

Dihydropteridine reductase (DHPR) catalyzes the NAD(P)H-mediated reduction of quinonoid dihydropteridine as a part of pterin-dependent aromatic amino acid hydroxylation. We isolated a fragment of Drosophila DHPR gene by PCR using degenerate primers. By screening a cDNA library, we obtained full-length clones. The predicted amino acid sequence of the Drosophila DHPR protein was highly homologous to other species including human and mouse. In particular, the Tyr-(Xaa)(3)-Lys motif, known as the NAD(P)H binding domain, and most amino acids relevant to quinonoid dihydropteridine binding site are identical to human DHPR. The recombinant DHPR protein expressed in Escherichia coli showed DHPR enzyme activity. Northern blot analysis revealed two transcripts of 1.1 and 0.9 kb. Genomic DNA sequencing revealed that the gene consists of two exons interrupted by a single 96-bp intron. The two transcripts have alternative promoters, both having no putative TATA box or CAAT box, but sharing a common poly(A)(+) signal. The existence of two alternative promoters suggests that each transcript be regulated independently through different stimuli. Further study is needed to examine the expression and function of the two alternative transcripts.

Amino Acid Sequence↗

PubMed2000

Signaling pathways to the assembly of an interferon-beta enhanceosome. Chemical genetic studies with a small molecule.

Small molecules that modulate specific protein functions are valuable tools for dissecting complex signaling pathways. Here, we identified a small molecule that induces the assembly of the interferon-beta (IFN-beta) enhanceosome by stimulating all the enhancer-binding activator proteins: ATF2/c-JUN, IRF3, and p50/p65 of NF-kappaB. This compound stimulates mitogen-activated protein kinase kinase kinase 1 (MEKK1), which is a member of a family of proteins involved in stress-mediated signaling pathways. Consistent with this, MEKK1 activates IRF3 in addition to ATF2/c-JUN and NF-kappaB for the assembly of the IFN-beta enhanceosome. MEKK1 activates IRF3 through the c-JUN amino-terminal kinase (JNK) pathway but not the p38 and IkappaB kinase (IKK) pathway. Taken together with previous observations, these results implicate that, for the assembly of an IFN-beta enhanceosome, MEKK1 can induce IRF3 and ATF2/c-JUN through the JNK pathway, whereas it can induce NF-kappaB through the IKK pathway. Thus, specific MEKK family proteins may be able to integrate some of multiple signal transduction pathways leading to the specific activation of the IFN-beta enhanceosome.

Enhancer Elements, Genetic↗

PubMed2000

Fringe forms a complex with Notch.

The Fringe protein of Drosophila and its vertebrate homologues function in boundary determination during pattern formation. Fringe has been proposed to inhibit Serrate-Notch signalling but to potentiate Delta-Notch signalling. Here we show that Fringe and Notch form a complex through both the Lin-Notch repeats and the epidermal growth factor repeats 22-36 (EGF22-36) of Notch when they are co-expressed. The Abruptex59b (Ax59b) and AxM1 mutations, which are caused by missense mutations in EGF repeats 24 and 25, respectively, abolish the Fringe-Notch interaction through EGF22-36, whereas the l(1)N(B) mutation in the third Lin-Notch repeat of Notch abolishes the interaction through Lin-Notch repeats. Ax mutations also greatly affect the Notch response to ectopic Fringe in vivo. Results from in vitro protein mixing experiments and subcellular colocalization experiments indicate that the Fringe-Notch complex may form before their secretion. These findings explain how Fringe acts cell-autonomously to modulate the ligand preference of Notch and why the Fringe-Notch relationship is conserved between phyla and in the development of very diverse structures.

Animals↗

PubMed2000

Identification of Mad as a repressor of the human telomerase (hTERT) gene.

Activation of telomerase, which has been frequently associated with cellular immortality, may constitute a key step in the development of human cancer. De-repression in the expression of its catalytic subunit hTERT gene has been proposed to directly link to the telomerase activation in tumor cells. Little is known about the mechanism how the hTERT gene is repressed in telomerase-negative mortal cells. This study was conducted, using an expression cloning approach, with the aim of identifying the gene(s) responsible for repressing the hTERT gene expression. Using this genetic screen, we isolated the transcription factor Mad as a repressor. Mutation of its DNA binding sites caused significant de-repression of hTERT promoter activity in mortal cells. This Mad-mediated repression of the hTERT promoter in mortal cells was counteracted by ectopic expression of Myc. The antagonism between Mad and Myc was also observed with an endogenous hTERT promoter. Their potential roles in differential hTERT promoter activities were further supported by the relative amounts of Mad and Myc proteins detected in immortal and mortal cells. Thus, Mad may be a direct negative regulator of hTERT in mortal cells and this repression mechanism can be inhibited by induction of Myc in immortal cells.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2000

Chemotherapeutic DNA-damaging drugs activate interferon regulatory factor-7 by the mitogen-activated protein kinase kinase-4-cJun NH2-terminal kinase pathway.

Chemotherapeutic drugs and energy-rich radiation cause DNA damage, inducing signaling pathways for apoptotic cell death or cell growth arrest. The tumor suppressor gene p53 plays the critical role in the regulation of these DNA damage responses. Human tumor cells can become resistant to chemotherapy through functional inactivation of p53. Thus, it is important to identify p53-independent DNA damage signaling pathways. Here, treatment of cells with chemotherapeutic drugs or UV irradiation potentiated the transcriptional activity of IFN regulatory factor-7 (IRF7), inducing its phosphorylation and its nuclear translocation. Furthermore, IRF7 was activated by the c-Jun NH2-terminal kinase (JNK) in response to DNA-damaging agents. Activation of JNK by mitogen-activated protein kinase kinase-4 stimulated the transcriptional activity of IRF7 and induced its translocation into the nucleus. Thus, activation of IRF7 through the JNK signaling pathway may play a role in the transcriptional regulation of genes in response to DNA-damaging agents.

Antineoplastic Agents↗

PubMed2000

Involvement of retinoblastoma protein in p27Kip1-induced apoptosis.

p27Kip1, a cyclin-dependent kinase (CDK) inhibitor, plays a critical role in cell cycle regulation. Expression of p27Kip1 is shown to increase during apoptosis in mammalian cells. Here, to directly address the role of p27Kip1 in apoptosis, p27Kip1 is overexpressed in human SK-Hep1 hepatoma cells. This leads to apoptotic cell death and this reduces protein, but not mRNA, levels of the retinoblastoma (Rb). Consistently, accumulation of Rb protein blocks p27Kip1-mediated apoptosis. These studies demonstrate an involvement of Rb in the apoptotic cell death which is induced by overexpression of p27Kip1.

Apoptosis↗

PubMed1999

The Wilms' tumor 1 tumor suppressor gene represses transcription of the human telomerase reverse transcriptase gene.

Regulation of the human telomerase reverse transcriptase (hTERT) gene is the primary determinant for telomerase enzyme activity, which is found in tumor cells but is largely absent from normal somatic cells. Recent studies have shown that Myc protein can transcriptionally activate the hTERT gene. However, little is known about the repression mechanism of the hTERT gene and telomerase enzyme. Here, we developed an expression cloning strategy to identify cDNAs whose products can repress hTERT promoter activity in telomerase-positive immortal cells. Using this screen, we isolated the Wilms' tumor 1 suppressor gene (WT1). WT1 can repress hTERT promoter activity in 293 kidney cells. The WT1 binding site on the hTERT promoter was identified by deletional analysis. Alteration of the WT1 binding site markedly derepresses transcription from an isolated hTERT promoter by inhibiting interaction of WT1 with DNA. These specific repression effects of WT1 were not observed in HeLa cells, which express no endogenous WT1. Furthermore, we show that WT1 can repress the endogenous hTERT promoter and telomerase enzyme activities. These results suggest that WT1 may be a transcriptional repressor of the hTERT gene, at least in some specific cells.

DNA-Binding Proteins↗

PubMed1999

Activation of interferon regulatory factor 3 in response to DNA-damaging agents.

Genotoxic stress triggers signal transduction pathways that mediate either the protection or apoptosis of affected cells. The interferon regulatory factors (IRFs) are involved in a wide range of host defense mechanisms against environmental stresses. Treatment with DNA-damaging agents, including doxorubicin and UV radiation, caused phosphorylation of the IRF3 transcription factor. Phosphorylation of IRF3 induced its interaction with the transcriptional co-activator cAMP-response element binding protein-binding protein. Furthermore, genotoxic stress-induced phosphorylation of IRF3 resulted in its movement from the cytoplasm to the nucleus, where it activated transcription from its binding site. These observations suggest that IRF3 plays a role in the defensive responses induced by genotoxic stress.

Amino Acid Substitution↗

PubMed1999

In vivo and in vitro analyses of Myc for differential promoter activities of the human telomerase (hTERT) gene in normal and tumor cells.

Induction of the catalytic subunit of human telomerase, hTERT, plays a critical role in the activation of telomerase during tumorigenesis. Here, we isolate the hTERT gene promoter and define the functional promoter region, which is inactive in normal cells but active in tumor cells. Myc directly interacts with the hTERT promoter and activates its transcription both in vivo and in vitro. Activation or repression of Myc can alter hTERT promoter activities in normal or tumor cells. Furthermore, we detect high levels of Myc protein in tumor cells compared with normal cells. The ability of Myc to modulate the activity of the hTERT promoter and, hence, the telomerase enzyme may contribute to its ability to promote tumor formation.

Base Sequence↗

PubMed1999

Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl tetrahydropterin synthase.

The enzyme 6-pyruvoyl tetrahydropterin synthase (PTPS) catalyses the second step in the de novo biosynthesis of tetrahydrobiopterin, the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. The Zn and Mg-dependent reaction includes a triphosphate elimination, a stereospecific reduction of the N5-C6 double bond and the oxidation of both side-chain hydroxyl groups. The crystal structure of the inactive mutant Cys42Ala of PTPS in complex with its natural substrate dihydroneopterinetriphosphate was determined at 1.9 A resolution. Additionally, the uncomplexed enzyme was refined to 2.0 A resolution. The active site of PTPS consists of the pterin-anchoring Glu A107 neighboured by two catalytic motifs: a Zn(II) binding site and an intersubunit catalytic triad formed by Cys A42, Asp B88 and His B89. In the free enzyme the Zn(II) is in tetravalent co-ordination with three histidine ligands and a water molecule. In the complex the water is replaced by the two substrate side-chain hydroxyl groups yielding a penta-co-ordinated Zn(II) ion. The Zn(II) ion plays a crucial role in catalysis. It activates the protons of the substrate, stabilizes the intermediates and disfavours the breaking of the C1'C2' bond in the pyruvoyl side-chain. Cys A42 is activated by His B89 and Asp B88 for proton abstraction from the two different substrate side-chain atoms C1', and C2'. Replacing Ala A42 in the mutant structure by the wild-type Cys by modelling shows that the C1' and C2' substrate side-chain protons are at equal distances to Cys A42 Sgamma. The basicity of Cys A42 may be increased by a catalytic triad His B89 and Asp B88. The active site of PTPS seems to be optimised to carry out proton abstractions from two different side-chain C1' and C2' atoms, with no obvious preference for one of them. Kinetic studies with dihydroneopterin monophosphate reveal that the triphosphate moiety of the substrate is necessary for enzyme specifity.

Animals↗

PubMed1999

Human cytomegalovirus IE1 protein activates AP-1 through a cellular protein kinase(s).

During human cytomegalovirus (HCMV) infection, a rapid increase in AP-1 activity is detected. In this study, activation of transcription from promoters containing AP-1-binding sites by the IE1 protein of HCMV was examined. In transient transfection assays with reporter plasmids, it was found that IE1 strongly induced AP-1-driven transcription. Cells stably expressing IE1 also showed higher levels of AP-1 activity than did control cells. IE1 expression did not raise levels of c-jun and c-fos RNA, as determined by quantitative RT-PCR. AP-1 induction by IE1 was blocked efficiently by protein kinase inhibitors in a cell type-dependent manner; for example, by staurosporine in the human microglial cell line U373MG and by H7 in the human promonocytic cell line U937. IE1-driven activation of AP-1 was increased dramatically by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1). The results of this study indicate that IE1 activates AP-1 at the post-transcriptional level and that MEKK1 may play an important role in this process.

Cells, Cultured↗

PubMed1998

An analysis of two tandem promoters of the Drosophila purple gene.

We have analyzed two tandem promoters, separated by only about 400 bp, of the purple (pr) gene of Drosophila melanogaster, by fusing them to the firefly luciferase reporter gene and employing a transient expression assay with Drosophila S2 cells. Both the distal promoter and the proximal promoter were found to function in S2 cells and an about 700 bp long region (-270 to +421), containing both promoters, was sufficient to effect maximal promoter activity. When the two promoters were analyzed separately, the distal promoter was found to be much stronger in its function than the proximal promoter. At least three different kinds of cis elements near the transcription start site appear to play crucial roles in driving constitutive expression from the distal promoter. On the other hand, only a single cis element, which may play a role in tissue-specific expression, appears to be important for the activity of the proximal promoter in S2 cells. We propose that the clustering of important cis elements near the transcription start sites may be responsible for the selective regulation of the two tandem promoters.

Animals↗

PubMed1998

Isolation and characterization of the Drosophila melanogaster cDNA encoding the sepiapterin reductase.

We have isolated and characterized the cDNA encoding Drosophila melanogaster sepiapterin reductase (SR). The amino acid sequence deduced from the cDNA sequence was 29% identical to those of mammalian SRs. The active site residues proposed from the three-dimensional structure of mouse SR are well conserved in Drosophila SR. The protein-coding region of the cDNA was expressed in Escherichia coli as a histidine fusion protein, and the resulting recombinant protein proved to have SR activity. The SR activity of the recombinant protein was inhibited by two indoleamines, N-acetyl serotonin and melatonin. Southern analysis suggests that the Drosophila SR gene is encoded by a single copy gene. RNA blot analysis revealed that the gene expresses 1.5 kb mRNA in both adult heads and bodies.

Alcohol Oxidoreductases↗

PubMed1998

Molecular characterization of the Drosophila melanogaster gene encoding the pterin 4alpha-carbinolamine dehydratase.

We have isolated and characterized the cDNA and the genomic DNA encoding Drosophila melanogaster pterin 4alpha-carbinolamine dehydratase (PCD). The amino acid sequence deduced from the cDNA sequence was very similar to those of PCDs previously reported in other species (19-57% identity). The protein coding region of the cDNA was expressed in E. coli as a histidine fusion protein, and the expressed protein proved to have PCD activity. The characterization of the Drosophila genomic clone revealed that the Drosophila PCD gene is interrupted by two introns. The potential promoter region, deduced from the determination of the transcription start point (tsp), lacks the distinct TATAAA box consensus sequence.

Amino Acid Sequence↗