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Sanggyu Lee

Publications and source records attributed to Sanggyu Lee.

17 recordsLinked to original sources
PubMed2006

The parthenogenetic activation of canine oocytes with Ca-EDTA by various culture periods and concentrations.

In the present study, canine oocytes were exposed to various concentrations of and durations of exposure to EDTA saturated with Ca(2+) (Ca-EDTA), a cell membrane-impermeable metal ion chelator, to determine if parthenogenetic activation could be induced. When oocytes were cultured for 48 or 72 h in parthenogenetic activation medium (PAM) without Ca-EDTA (control) or PAM supplemented with 1 or 5mM Ca-EDTA, the highest rate of pronuclear formation (PN) was obtained in oocytes cultured in 1mM Ca-EDTA for 48 h (8.0%; P<0.05). There was no pronuclear formation in the control group (PAM without Ca-EDTA). Oocytes treated with 5mM Ca-EDTA for 48 h or 1mM Ca-EDTA for 72 h formed a parthenogenetic pronucleus (3.1 and 4.5, respectively). However, there was no pronuclear formation in oocytes treated with 5mM Ca-EDTA for 72 h. In summary, exposure to Ca-EDTA can induce pronuclear formation in canine oocytes.

Animals↗
PubMed2006

Gene expression profiles in acute myeloid leukemia with common translocations using SAGE.

Identification of the specific cytogenetic abnormality is one of the critical steps for classification of acute myeloblastic leukemia (AML) which influences the selection of appropriate therapy and provides information about disease prognosis. However at present, the genetic complexity of AML is only partially understood. To obtain a comprehensive, unbiased, quantitative measure, we performed serial analysis of gene expression (SAGE) on CD15(+) myeloid progenitor cells from 22 AML patients who had four of the most common translocations, namely t(8;21), t(15;17), t(9;11), and inv(16). The quantitative data provide clear evidence that the major change in all these translocation-carrying leukemias is a decrease in expression of the majority of transcripts compared with normal CD15(+) cells. From a total of 1,247,535 SAGE tags, we identified 2,604 transcripts whose expression was significantly altered in these leukemias compared with normal myeloid progenitor cells. The gene ontology of the 1,110 transcripts that matched known genes revealed that each translocation had a uniquely altered profile in various functional categories including regulation of transcription, cell cycle, protein synthesis, and apoptosis. Our global analysis of gene expression of common translocations in AML can focus attention on the function of the genes with altered expression for future biological studies as well as highlight genes/pathways for more specifically targeted therapy.

Apoptosis↗
PubMed2005

2.45 GHz radiofrequency fields alter gene expression in cultured human cells.

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.

Dose-Response Relationship, Radiation↗
PubMed2005

Stage-dependent gene expression profiles during natural killer cell development.

Natural killer (NK) cells develop from hematopoietic stem cells (HSCs) in the bone marrow. To understand the molecular regulation of NK cell development, serial analysis of gene expression (SAGE) was applied to HSCs, NK precursor (pNK) cells, and mature NK cells (mNK) cultured without or with OP9 stromal cells. From 170,464 total individual tags from four SAGE libraries, 35,385 unique genes were identified. A set of genes was expressed in a stage-specific manner: 15 genes in HSCs, 30 genes in pNK cells, and 27 genes in mNK cells. Among them, lipoprotein lipase induced NK cell maturation and cytotoxic activity. Identification of genome-wide profiles of gene expression in different stages of NK cell development affords us a fundamental basis for defining the molecular network during NK cell development.

Animals↗
PubMed2005

Serial analysis of gene expression study of a hybrid rice strain (LYP9) and its parental cultivars.

Using the serial analysis of gene expression technique, we surveyed transcriptomes of three major tissues (panicles, leaves, and roots) of a super-hybrid rice (Oryza sativa) strain, LYP9, in comparison to its parental cultivars, 93-11 (indica) and PA64s (japonica). We acquired 465,679 tags from the serial analysis of gene expression libraries, which were consolidated into 68,483 unique tags. Focusing our initial functional analyses on a subset of the data that are supported by full-length cDNAs and the tags (genes) differentially expressed in the hybrid at a significant level (P<0.01), we identified 595 up-regulated (22 tags in panicles, 228 in leaves, and 345 in roots) and 25 down-regulated (seven tags in panicles, 15 in leaves, and three in roots) in LYP9. Most of the tag-identified and up-regulated genes were found related to enhancing carbon- and nitrogen-assimilation, including photosynthesis in leaves, nitrogen uptake in roots, and rapid growth in both roots and panicles. Among the down-regulated genes in LYP9, there is an essential enzyme in photorespiration, alanine:glyoxylate aminotransferase 1. Our study adds a new set of data crucial for the understanding of molecular mechanisms of heterosis and gene regulation networks of the cultivated rice.

Chromosome Mapping↗
PubMed2005

Detecting novel low-abundant transcripts in Drosophila.

Increasing evidence suggests that low-abundant transcripts may play fundamental roles in biological processes. In an attempt to estimate the prevalence of low-abundant transcripts in eukaryotic genomes, we performed a transcriptome analysis in Drosophila using the SAGE technique. We collected 244,313 SAGE tags from transcripts expressed in Drosophila embryonic, larval, pupae, adult, and testicular tissue. From these SAGE tags, we identified 40,823 unique SAGE tags. Our analysis showed that 55% of the 40,823 unique SAGE tags are novel without matches in currently known Drosophila transcripts, and most of the novel SAGE tags have low copy numbers. Further analysis indicated that these novel SAGE tags represent novel low-abundant transcripts expressed from loci outside of currently annotated exons including the intergenic and intronic regions, and antisense of the currently annotated exons in the Drosophila genome. Our study reveals the presence of a significant number of novel low-abundant transcripts in Drosophila, and highlights the need to isolate these novel low-abundant transcripts for further biological studies.

Animals↗
PubMed2004

SAGE is far more sensitive than EST for detecting low-abundance transcripts.

BACKGROUND: Isolation of low-abundance transcripts expressed in a genome remains a serious challenge in transcriptome studies. The sensitivity of the methods used for analysis has a direct impact on the efficiency of the detection. We compared the EST method and the SAGE method to determine which one is more sensitive and to what extent the sensitivity is great for the detection of low-abundance transcripts. RESULTS: Using the same low-abundance transcripts detected by both methods as the targeted sequences, we observed that the SAGE method is 26 times more sensitive than the EST method for the detection of low-abundance transcripts. CONCLUSIONS: The SAGE method is more efficient than the EST method in detecting the low-abundance transcripts.

3' Untranslated Regions↗
PubMed2004

Analysis of translocations that involve the NUP98 gene in patients with 11p15 chromosomal rearrangements.

The NUP98 gene has been reported to be fused with at least 15 partner genes in leukemias with 11p15 translocations. We report the results of screening of cases with cytogenetically documented rearrangements of 11p15 and the subsequent identification of involvement of NUP98 and its partner genes. We identified 49 samples from 46 hematology patients with 11p15 (including a few with 11p14) abnormalities, and using fluorescence in situ hybridization (FISH), we found that NUP98 was disrupted in 7 cases. With the use of gene-specific FISH probes, in 6 cases, we identified the partner genes, which were PRRX1 (PMX1; in 2 cases), HOXD13, RAP1GDS1, HOXC13, and TOP1. In the 3 cases for which RNA was available, RT-PCR was performed, which confirmed the FISH results and identified the location of the breakpoints in patient cDNA. Our data confirm the previous findings that NUP98 is a recurrent target in various types of leukemia.

Adolescent↗
PubMed2003

T lymphoid differentiation in human bone marrow.

The unique role of the thymus in the development of T cells was established >4 decades ago. To elucidate how uncommitted lymphoid progenitor cells are instructed to migrate from bone marrow to the thymus to undergo T lymphoid differentiation, we generated and analyzed a genome-wide gene expression profile of CD7+ CD10+ human bone marrow T cell lineage precursors (TLPs) by using the serial analysis of gene expression technique. Unexpectedly, the serial analysis of gene expression profile identified a high number of (pre-) T cell receptor antigen (TCR)-related transcripts in bone marrow TLPs. To determine the configuration of the TCRbeta locus in these cells at a quantitative level, we sorted and analyzed bone marrow TLPs from five donors by single-cell PCR. Similar proportions of TLPs harbored TCRbeta germ-line alleles, D-J, or V-DJ gene rearrangements. Thus, bone marrow TLPs are heterogenous with respect to TCRbeta rearrangement status, suggesting an active recombination machinery that is consistent with the expression of RAG1, RAG2, and TdT in this population. As a hallmark of ongoing TCRbeta V-DJ rearrangement, we could amplify broken-ended recombination-signal sequence DNA intermediates from bone marrow TLPs, but not from mature T cells by ligation-mediated PCR. Approximately half of the TCRbeta rearrangements were compatible with the expression of a functional pre-TCR, which is in agreement with surface expression of pre-Talpha on bone marrow TLPs as shown by confocal laser microscopy and flow cytometry. At a frequency <0.5% of mononucleated cells in human bone marrow, this population is rare, yet it exemplifies T lymphoid differentiation in the human already before immigration into the thymus.

Base Sequence↗
PubMed2002

Silencing of B cell receptor signals in human naive B cells.

To identify changes in the regulation of B cell receptor (BCR) signals during the development of human B cells, we generated genome-wide gene expression profiles using the serial analysis of gene expression (SAGE) technique for CD34(+) hematopoietic stem cells (HSCs), pre-B cells, naive, germinal center (GC), and memory B cells. Comparing these SAGE profiles, genes encoding positive regulators of BCR signaling were expressed at consistently lower levels in naive B cells than in all other B cell subsets. Conversely, a large group of inhibitory signaling molecules, mostly belonging to the immunoglobulin superfamily (IgSF), were specifically or predominantly expressed in naive B cells. The quantitative differences observed by SAGE were corroborated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B cells at least partly results from interleukin (IL)-4 receptor signaling. Conversely, activation or impairment of the inhibitory IgSF receptor LIRB1 affected BCR-dependent Ca(2+) mobilization only in naive but not memory B cells. Thus, LIRB1 and IL-4 may represent components of two nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity maturation. In memory B cells, facilitated responsiveness upon reencounter of the immunizing antigen may result from amplification of BCR signals at virtually all levels of signal transduction.

Antigens, CD34↗
PubMed2002

Identifying novel transcripts and novel genes in the human genome by using novel SAGE tags.

The number of genes in the human genome is still a controversial issue. Whereas most of the genes in the human genome are said to have been physically or computationally identified, many short cDNA sequences identified as tags by use of serial analysis of gene expression (SAGE) do not match these genes. By performing experimental verification of more than 1,000 SAGE tags and analyzing 4,285,923 SAGE tags of human origin in the current SAGE database, we examined the nature of the unmatched SAGE tags. Our study shows that most of the unmatched SAGE tags are truly novel SAGE tags that originated from novel transcripts not yet identified in the human genome, including alternatively spliced transcripts from known genes and potential novel genes. Our study indicates that by using novel SAGE tags as probes, we should be able to identify efficiently many novel transcripts/novel genes in the human genome that are difficult to identify by conventional methods.

Base Pair Mismatch↗
PubMed2002

Molecular portraits of B cell lineage commitment.

In an attempt to characterize early B cell development including the commitment of progenitor cells to the B cell lineage, we generated and compared genomewide gene expression profiles of human hematopoietic stem cells (HSCs) and pre-B cells (PBCs) by using serial analysis of gene expression. From more than 100,000 serial analysis of gene expression tags collected from human CD34(+) HSCs and CD10(+) CD19(+) PBCs, 42,399 unique transcripts were identified in HSCs but only 16,786 in PBCs, suggesting that more than 60% of transcripts expressed in HSCs were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptor (pre-BCR)-associated genes are virtually missing in HSCs but account for more than 10% of the transcriptome of PBCs, which also show increased expression of apoptosis-related genes. Both concentration of the transcriptional repertoire on pre-BCR-related genes together with marked up-regulation of apoptosis mediators in PBC might reflect selection for the expression of a functional pre-BCR within the bone marrow. Besides known regulator genes of early B cell development such as PAX5, E2A, and EBF, the most abundantly expressed genes in PBCs include ATM, PDGFRA, SIAH1, PIM2, C/EBPB, WNT16, and TCL1, the role of which has not been established yet in early B cell development.

Antigens, CD↗
PubMed2002

Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription.

We have analyzed a systematic flaw in the current system of gene identification: the oligo(dT) primer widely used for cDNA synthesis generates a high frequency of truncated cDNAs through internal poly(A) priming. Such truncated cDNAs may contribute to 12% of the expressed sequence tags in the current dbEST database. By using a synthetic transcript and real mRNA templates as models, we characterized the patterns of internal poly(A) priming by oligo(dT) primer. We further demonstrated that the internal poly(A) priming can be effectively diminished by replacing the oligo(dT) primer with a set of anchored oligo(dT) primers for reverse transcription. Our study indicates that cDNAs designed for genomewide gene identification should be synthesized by use of the anchored oligo(dT) primers, rather than the oligo(dT) primers, to diminish the generation of truncated cDNAs caused by internal poly(A) priming.

Animals↗
PubMed2002

High-throughput GLGI procedure for converting a large number of serial analysis of gene expression tag sequences into 3' complementary DNAs.

Serial analysis of gene expression (SAGE) is a powerful technique for genome-wide analysis of gene expression. However, two-thirds of SAGE tags cannot be used directly for gene identification for two reasons. First, many SAGE tags match several known expressed sequences, owing to the short length of SAGE tag sequences. Second, many SAGE tags do not match any known expressed sequences, presumably because the sequences corresponding to these SAGE tags have not been identified. These two problems can be solved by extension of the SAGE tags into 3' complementary DNAs (cDNAs) by use of the GLGI technique (generation of longer cDNA fragments from SAGE tags for gene identification). We have improved the original GLGI technique into a high-throughput procedure for simultaneous conversion of a large number of SAGE tags into corresponding 3' cDNAs. The whole process is simple, rapid, low-cost, and highly efficient, as shown by our use of this procedure for analyzing hundreds of SAGE tags. In addition to identifying the correct gene for SAGE tags with multiple matches, GLGI can be used for large-scale identification of novel genes by converting novel SAGE tags into 3' cDNAs. Applying this high-throughput procedure should accelerate the rate of gene identification significantly in the human and other eukaryotic genomes.

Animals↗
PubMed2002

Correct identification of genes from serial analysis of gene expression tag sequences.

SAGE (serial analysis of gene expression) is a remarkable technique for genome-wide analysis of gene expression. It is crucial to understand the extent to which SAGE can accurately indicate a gene or expressed sequence tag (EST) with a single tag. We analyzed the effect of the size of SAGE tag on gene identification. Our observation indicates that SAGE tags are in general not long enough to achieve the degree of uniqueness of identification originally envisaged. Our observations also indicate that the limitation of using SAGE tag to identify a gene can be overcome by converting SAGE tags into longer 3' EST sequences with the generation of longer cDNA fragments from SAGE tages for gene identification (GLGI) method.

Expressed Sequence Tags↗
PubMed2002

Computational Analysis of Gene Identification with SAGE.

SAGE is one of the few techniques capable of uniformly probing gene expression at a genome level irrespective of mRNA abundance and without a priori knowledge of the transcripts present. However, individual SAGE tags can match many sequences in the reference database, complicating gene identification. We perform a baseline evaluation of gene identification with SAGE using UniGene Human as the reference database by analyzing 1) the distributions of tags for various length tag sets formed for UniGene Human and 2) the tag-to-sequence mapping using a SAGE tag set consisting of 37,522 tags derived from human myeloid cells. The extensive multiplicity of the dbEST component of UniGene significantly detracts from gains that might be expected by extending tags within the scope of the SAGE protocol. In order to achieve reasonable sequence specificity for gene identification with the content of the commonly used UniGene sequence collection, tags on the order of hundreds of bases in length are required. One way to produce tags of such lengths is with GLGI, which extends SAGE tags to the 3' end of cDNA. We show that the longer sequences produced by GLGI relieve significantly the multiple match condition. In the myeloid sample, we also found a correlation between multiple match severity and high copy number. We extrapolate these findings, providing insights into the use of UniGene Human as a reference for gene identification.

Animals↗