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FLJ39061 new gene expression in lymphoma and Functional Analysis_1474

By Alfred Berry,2014-10-30 17:51
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FLJ39061 new gene expression in lymphoma and Functional Analysis_1474

    FLJ39061 new gene expression in lymphoma and Functional Analysis

     Author: Yan Lingling, Zhu Tao, Xiang-Yang Bai, Yun-

    Ping Lu, Jian-Feng Zhou, Martin

     Abstract Objective To study the proliferation of lymphoma and reactive lymph nodes differences in gene expression, and high expression of genes FLJ39061 lymphoma preliminary analysis. Methods lymphoma and reactive hyperplasia of lymph node specimens of liquid nitrogen frozen lymph node biopsy specimens, the use of laser microdissection separation of

    lymphocytes and lymphocytes in the mRNA extracted and cDNA microarray hybridization, scanning through the signal after treatment to obtain the expression of two differences in genes. Using bioinformatics methods a significant difference

    between the preliminary analysis of new gene FLJ39061. The results of cDNA microarray identified 152 differentially expressed genes. Lymphoma-related gene FLJ39061 on the nature of effective prediction, the basic protein encoded by the gene identified as a nuclear protein, is likely to be one MAP kinase-activated protein kinase 2, precursors or isomers. Conclusion FLJ39061 may be used as kinase substrate phosphorylation regulates cell metabolism, and lymphoma related to the occurrence can be used as therapeutic targets

    for further functional studies.

     Key words lymphoma; cDNA microarray; FLJ39061; bio-

    informatics; MAPK

     Expression of a Novel Gene FLJ39061 in Lymphoma and Its Function Analysis

     Abstract: Objective To explore the differential expressed

    genes in lymphoma and reactive lymph node and performed an initial analysis on a novel gene. Hypethetical Protein FIJ39061, which is highly expressed in lymphnode of lymphoma. Methods The samples were collected and flash frozen quickly to

    maintain the integrity of mRNA. The mRNA of the tissue from the lymphoma and reactive lymph node are marked with biotin respectively and hybridized with expression profile microarray, and the differential expressed genes are obtained. Initial bioinformatics analysis is performed on a novel gene named FLJ39061 which have no functional researches at present. Its gene structure, genomic localization, the physical and chemical characteristics of the putative protein, subcellular localization, functional domain and so on are predicted, and

    the systematic evolution analysis is performed on the similar proteins among several species. Results Using expression profile microarray, 152 differential expressed genes are uncovered. Efficient bioinformatics analysis have fundamentally identified that FLJ39061 is a nuclear protein, and probably is precursor or isomer of MAP kinase activated

    protein kinase 2. Conclusion By bioinformatics analysis, FLJ39061 may involved in the phosphorylation regulation. It is anticipated that this project will advance our understanding of the molecular mechanisms in the neoplastic transformation of lymphoma and the new insights will help to identify promising molecular targets for therapeutic intervention.

     Key words: Lymphoma; Expression profile microarray;

    FLJ39061; Bioinformatics; MAPK

     0 Introduction

     In recent years, there are incremental trend in the incidence of malignant lymphoma, and its pathogenesis is still unclear. With the gene chips, serial analysis of gene expression (SAGE), suppression subtractive hybridization (SSH) the development of high-throughput research methods, making

    the molecular level to study the regulation of tumor-related

    molecular mechanisms and regulatory pathways. Here the use of gene chips for laser capture microdissection to obtain gene expression profiles of lymphoma lymph node samples for analysis and found a T-cell lymphoma in the lymph nodes in the highly expressed genes FLJ39061, and its preliminary bioinformatics analysis, the basic clear The biological

    function of this gene and significance for the future of

    molecular biology experiments provided further information and methodological guidance.

     1 Materials and methods

     1.1 Materials

     6 cases of lymphoma and reactive hyperplasia of lymph

    node specimens from lymph node specimens, Huazhong University of Science and Technology, Tongji Hospital, Tongji Medical College in September 2005 ~ December 2005 hematology patients surgical specimens, including three pathologic findings

    confirmed the lymphoma (2, called B-cell lymphoma, a known T-

    cell lymphoma) and the other three patients with pathological lymph nodes were identified as reactive hyperplasia. Micro mRNA extraction reagents were purchased from Qiagen Company. CDNA microarray biochip companies using Affymetrix Human Genome U133 plus2.0 chips. The chip covers about 54 000 individual human gene sequences.

     1.2 Methods

     1.2.1 Total RNA extraction and purification of

     Fresh lymph node tissue samples drawn after the bacteria

    stored in cold fixative in the back lab. Frozen sections will be organized, laser capture micro-lymphoma cells, micro-

    extraction of cells in the total RNA.

     1.2.2 Micro-RNA in vitro amplification of

     Using the T7 OligodT do reverse reaction, synthesis of

    double-stranded cDNA with T7 RNA polymerase in vitro

    transcription to do. unbiased linear amplification of mRNA for the future, with 1% agarose gel electrophoresis and UV spectrophotometer obtained aRNA purity and concentration.

     1.2.3 cDNA microarray sample preparation and detection

     After aRNA amplification carried out on the quality of identification, in order to biotin incorporation of reverse transcription, labeling aRNA. Followed by microarray hybridization.

     1.2.4 cDNA microarray analysis of raw data

     Gene chip every gene or EST from the 11 20 to 25 base

    pairs of the probe composition. Hybridization signal data measured after deducting background values, using Wilcoxon signed-rank test probe hybridization signal intensity and the corresponding genes in control mismatch probe differences. To

    0.05 as a significant difference in the standard. P ? 0.05 of

    the gene as a positive gene. Comprehensive comparison of test results 6 times and returned to lymphoma and reactive hyperplasia of lymph nodes differentially expressed sequence information.

     1.2.5 Bioinformatic analysis

     Sequence of reference sequence and the basic notes from NCBI's GenBank database, using the genome map viewer watching location and gene structure. The use of Spidey Tools (http://www.ncbi.nlm.nih.gov/IEB/Research / Ostell / Spidey /)

    will be aligned to the corresponding mRNA sequence of the genome sequence to verify the gene structure. Expasy site (http://www.expasy.org/) of the ProtParam tool to predict physical and chemical properties of proteins, ProtScale tool

    to predict hydrophilicity profile. InterProScan [2] (http://www.ebi.ac.uk/InterProS can /) prediction of

    protein functional domains and structural domains. PSORT II (http://psort.nibb.ac.jp/form2.html) predicted subcellular location information. SignalP3.0 Server

    (http://www.cbs.dtu.dk/services/SignalP/) predicted a possible signal peptide cleavage site [3]. TMHMM Server

    (http://www.cbs.dtu.dk/services/TMHMM/) predicted transmembrane. Motif Scan (http://myhits.isb sib.ch / cgi

     bin / motif_sc an) search for known functional motif. ELM [4] (http://elm.eu.org/) prediction of protein functional sites. Search NCBI's UniGene

    (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene) understanding of tissue expression of protein abundance and expression of the abundance of different developmental stages. Using the BlastP tool to query the NCBI non-redundant protein

    database to find out whether other species have similar protein exists. ClustalX software for protein multiple sequence alignment, and build evolutionary trees. Using

    Bootstrap method generates a random seed from the test 1000 times to verify the reliability of evolutionary trees.

TreeView software observe evolutionary tree.

     2 Results

     2.1 Identification of tissue integrity and RNA extraction

    results

     HE staining after the Organization of frozen sections, laser capture micro-lymph node lymphocytes. Extraction of

    micro-RNA, in vitro amplified RNA integrity of the test results showed that the 18S, 28S RNA bands clear, appropriate

    proportion, RNA integrity of the good.

     2.2 cDNA microarray results

     According to chip provided by cDNA microarray results, in order to ensure sufficient specificity of the differential gene selection criteria: the 0.05 significance for the

    difference in criteria for judging the performance of 3 consecutive lymphoma, positive, while in reactive hyperplasia 3 times the performance of continuous negative gene as a candidate gene. According to the screening criteria, lymphoma increase in the expression of the nucleotide sequences 80, 13 subordinated to the known genes, no sequences related to known genes 67; expression of the nucleotide sequences reduced 72, subordinate to the known gene sequence are 26, no related sequences of known genes 46. These genes are most likely to

    have significant biological study of genes, there are 15. Hypothetical Protein FLJ39061 which is a new protein, yet research coverage of relevant literature. Therefore, the nucleotide sequences of FLJ39061 further bioinformatics

    analysis. Reposted elsewhere in the paper for free download http://

     2.3 Bioinformatics analysis

     FLJ39061 length of 2492 bp, CDS at 315 ~ 2492 bp, encoding a 725aa protein XP_092342. MegaBlast tool to use the reference sequence XM_092342 gene FLJ39061 on the human genome database search, found at human chromosome 2q33.1 of the positive-strand, MapViewer can be very intuitive to see that the gene has 14 exons and 13 introns , shown in Figure 1. Spidey has also confirmed the result using the XM_092342

    sequence and genomic sequence NC_000002 conducted in

    conjunction with, the 14 exons and the genomic sequences matching the consistency of 100%, and the tips of all exons were observed for a typical position-point cut and shear

    receptor site. AK096380 sequence is incomplete and contains a coding region of cDNA clones. AK096380 and XM_092342 for the nucleic acid sequences and encoded protein sequence of both comparisons. Compared with the results shown in Figure 2. The results showed that, AK096380 almost all over the previous

    XM_092342 than the situation for the Identities = 2110/2113 (99%), remove the three point mutations, this is basically that the two are matched in the AK096380 length, and the two is very close to matching the length of the first 11 exons

    length. In addition, the comparison between the two protein sequences result Identities = 547/582 (93%), Positives = 547/582 (93%). Which illustrates, AK096380 may be part of the sequence of XM_092342, or variants of its cut, only the former sequence, there are several point mutations, resulting in protein-coding is slightly different. And AK096380 sequence does not terminate the password, may not be the full-length

    mRNA reverse transcription product.

     The gene FLJ39061 protein reference sequence XP_092342

    submitted to the ProParam, predicted molecular mass of 81,253.5 and theoretical isoelectric point of 9.16, for a basic macromolecules protein. Half-life in vitro human

    reticulocytes as 30h, instability index 60.59, was classified as unstable proteins. ProScale reference Hphob. / Kyte

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