The thymus is the central lymphoid organ for the development of bone marrow-derived precursor cells into mature T-cells. Understanding the molecular mechanism of thymic involution and regeneration is critical to develop methods to normalize or improve host immunity from the decreased immune function caused by thymic involution. In this study, the regenerating thymus cDNA library was constructed in the rat from a model of thymic involution and regeneration induced by cyclophosphamide. Expressed sequence tags (ESTs) were obtained by partial sequencing of 700 randomly selected insert-containing clones. A total of 630 ESTs were analyzed, of which 486 ESTs (78%) matched to known genes and 125 ESTs (19%) matched to other ESTs (unknown genes). The 19 ESTs (3%) did not match with any known sequences. The ESTs were grouped into six main functional categories: metabolism (44%), signaling components (20%), membrane transport (7%), cytoskeleton (2%), cell division (2%) and defense (2%). As a result of RT-PCR analysis, expression of putative gene 01, putative E2IG2 gene, musculin and osteoactivin significantly increased in rat thymus during regeneration. The putative gene 01 showed complete homology with mitochondrial ribosomal protein S4 by homology search and multiple alignment of amino acid. These results provide the extensive molecular information on thymus regeneration and will be useful source to identify various genes which may play an important role in the thymus regeneration as well as to clone novel genes. Furthermore, the availability of these data will serve as a basis for further research to understand the molecular mechanism of thymus regeneration.
Animals ; Cell Division ; Clone Cells ; Cyclophosphamide* ; Cytoskeleton ; Expressed Sequence Tags* ; Gene Expression ; Gene Library ; Membranes ; Metabolism ; Rats* ; Regeneration* ; Ribosomal Proteins ; T-Lymphocytes ; Thymus Gland*

Long intergenic non-coding RNAs (lincRNAs) have historically been ignored in cancer biology. However, thousands of lincRNAs have been identified in mammals using recently developed genomic tools, including microarray and high-throughput RNA sequencing (RNA-seq). Several of the lincRNAs identified have been well characterized for their functions in carcinogenesis. Here we performed RNA-seq experiments comparing gastric cancer with normal tissues to find differentially expressed transcripts in intergenic regions. By analyzing our own RNA-seq and public microarray data, we identified 31 transcripts, including a known expressed sequence tag, BM742401. BM742401 was downregulated in cancer, and its downregulation was associated with poor survival in gastric cancer patients. Ectopic overexpression of BM742401 inhibited metastasis-related phenotypes and decreased the concentration of extracellular MMP9. These results suggest that BM742401 is a potential lincRNA marker and therapeutic target.
Animals ; DNA, Intergenic/genetics ; Expressed Sequence Tags/*metabolism ; Extracellular Space/metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Genetic Predisposition to Disease ; Humans ; Male ; Matrix Metalloproteinase 9/metabolism ; Mice ; Mice, Inbred C57BL ; Multivariate Analysis ; Neoplasm Metastasis ; Neoplasm Staging ; Phenotype ; Proportional Hazards Models ; RNA, Long Noncoding/*genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Reproducibility of Results ; Stomach Neoplasms/*genetics/*pathology ; Survival Analysis

Chinese agarwood is formed in the aromatic resinous wood formed in Aquilaria sinensis (Lour.) Gilg (botanical family: Thymelaeaceae). Only when suffering stress of wound, etc, can A. sinensis produce sesquiterpenes etc. compounds of agarwood around wounds. However, little is known about how wound induced the biosynthesis pathway of sesquiterpenes. To reveal the molecular mechanism of wound-induced agarwood formation, RNA sequencing (RNA-seq) technology was used to investigate the profile of gene expression in A. sinensis treated by mechanical wounding and elucidate its functional gene. A total of 40,295 ESTs with an average read length of 305 bp were generated and 22 095 unigenes were formed by initial gene splicing. 61.6% of these unigenes (13 611) were annotated using BLAST searches against the SwissProt, KEGG, Nr and Nt databases. Twenty-six unigenes (encoding 7 enzymes) were found to be involved in sesquiterpene of agarwood biosynthesis by bioinformatic tools of Gene Ontology and KEGG. Novel genes that are potentially involved in sesquiterpenes biosynthesis were identified in A. sinensis, providing data for further sesquiterpenes biosynthesis pathway by molecular methods and the EST data establish a foundation for future studies in the molecular mechanisms of wound-induce agarwood formation in A. sinensis.
Drugs, Chinese Herbal ; chemistry ; metabolism ; Expressed Sequence Tags ; Genes, Plant ; genetics ; Plants, Medicinal ; chemistry ; genetics ; Sequence Analysis, RNA ; Sesquiterpenes ; chemistry ; metabolism ; Stress, Physiological ; genetics ; Thymelaeaceae ; chemistry ; genetics ; Transcriptome ; genetics ; Wood ; genetics ; metabolism

Squalene synthase (SQS) is a key enzyme in plant terpenoid biosynthetic pathway. This study focused on cloning and analysis of Huperzia serrata SQS (HsSQS1) gene. After searching the transcriptome dataset of H serrata, one unique sequence encoding SQS was discovered. The primers were designed according to the transcript sequence of HsSQS1 from the H. serrata transcriptome dataset. The open reading frame of HsSQS1 was cloned using RT-PCR strategy. The bioinformatic analysis of this gene and its corresponding protein were performed. The cDNA (named as HsSQS1) contains a 1263 bp open reading frame and encodes a predicted protein of 420 amino acids. The GenBank accession number for this gene is JQ004938. HsSQS1 contains two transmembrane regions, without signal peptide. The conserved domain of squalene synthase was presented in HsSQS1. HsSQS1 was more abundant in H. serrata root than in leaf and stem. This study cloned and analyzed squalene synthase gene from H. serrata for the first time. The result will provide a foundation for exploring the mechanism ofterpenoid biosynthesis in H. serrata plants.
Amino Acid Sequence ; Biosynthetic Pathways ; Cloning, Molecular ; DNA, Complementary ; genetics ; Expressed Sequence Tags ; Farnesyl-Diphosphate Farnesyltransferase ; genetics ; isolation & purification ; metabolism ; Genes, Plant ; genetics ; Huperzia ; enzymology ; genetics ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; Plant Leaves ; enzymology ; Plant Roots ; enzymology ; Plant Stems ; enzymology ; Plants, Medicinal ; enzymology ; genetics ; Triterpenes ; chemistry

OBJECTIVETo explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monoculture Rehmannia glutinosa.

METHODTo use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony screening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics.

RESULTThe subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 (forward library) and 214 (reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, involving 21 metabolic pathways.

CONCLUSIONThe information of differential expression genes in continuous monoculture R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monoculture has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monoculture R. glutinosa.

Expressed Sequence Tags ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Gene Library ; Polymerase Chain Reaction ; Rehmannia ; genetics ; metabolism ; Sequence Analysis, DNA

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. To examine the gene expression profile in calcium-induced keratinocyte differentiation, we constructed a normalized cDNA library using mRNA isolated from these calcium-treated keratinocytes. After sequencing about 10,000 clones, we were able to obtain 4,104 independent genes. They consisted of 3,699 annotated genes and 405 expressed sequence tags (ESTs). Some were the genes involved in constituting epidermal structures and others were unknown genes that are probably associated with keratinocytes. In particular, we were able to identify genes located at the chromosome 1q21, the locus for the epidermal differentiation complex, and 19q13.1, another probable locus for epidermal differentiation-related gene clusters. One EST located at the chromosome 19q13.1 showed increased expression by calcium treatment, suggesting a novel candidate gene relevant to keratinocyte differentiation. These results demonstrate the complexity of the transcriptional profile of keratinocytes, providing important clues on which to base further investigations of the molecular events underlying keratinocyte differentiation.
Calcium/*metabolism ; Cells, Cultured ; Chromosome Mapping ; Chromosomes, Human ; Expressed Sequence Tags ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Library ; Humans ; Keratinocytes/cytology/*physiology ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis

OBJECTIVETo clone the mouse testis specific gene TSEG-2 via a bioinformatic approach.

METHODSThe expressed sequence tags (EST) in the normal mouse testis were obtained from the online EST database ZooDDD. Their highly homologous EST sequences were retrieved through the dbEST database to construct contigs and spliced with the biomedical software Biolign. The corresponding exons and introns within the genome sequences were predicted with the software GeneScan. Primers were designed according to the open reading frame. RT-PCR was applied in cloning the cDNA of the novel gene from the mouse testis tissue and analyzing its expression patterns in the undescended testis and various organ tissues as well as in different developmental stages of the mouse testis. The sequencing results of TSEG-2 underwent bioinformatic analyses.

RESULTSThe novel mouse testis gene TSEG-2 was successfully cloned, with full-length sequence of 451 bp. The open reading frame was 267 bp, coding a protein of 88 amino acid residues, and demonstrated to be correct by RT-PCR. The expression of TSEG-2 was high in the mouse testis, regular in the testis cDNA samples of different postnatal days, and down-regulated in the cryptorchidism model. No obvious homology with other mouse cDNA was found for TSEG-2. The GenBank accession number EU079025 was achieved. Function prediction showed that mouse TSEG-2 was probably a soluble non-secretary protein located at chromosome 15qE3, or a nucleoprotein with 2 phosphorylation sites of protein kinase C (PKC) and 1 of casein kinase II (CK2).

CONCLUSIONA novel mouse testis specific gene TSEG-2 was successfully cloned, which could be down-regulated by cryptorchidism-inducible 17-beta estradiol. This has prepared the ground for further researches on the biological function and expression regulation of TSEG-2.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Expressed Sequence Tags ; Female ; Gene Expression ; Male ; Mice ; Mice, Inbred Strains ; Molecular Sequence Data ; Open Reading Frames ; Pregnancy ; Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Testis ; metabolism

OBJECTIVETo screen differential expression genes and proteins at transcriptome and proteome levels between human gastric cancer tissue and corresponding normal mucosa.

METHODSFresh-frozen gastric cancers were collected from patients treated at Ruijin Hospital. A total of 22 pairs of gastric cancer tissues and the corresponding noncancerous mucosa were analyzed. Commercially available cDNA microarray with 14 592 genes/ESTs was used. Genes were considered to be up-or down-regulated when the intensity ratio Cy3/Cy5 was > or = 2 or < or = 0.5 in over 50% samples (P<0.05). Immobilized pH gradient(IPG)-based 2-DE was applied to separate the total proteins of gastric cancer tissue and paired normal tissue. After staining and analysis by software,the differential expression proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS.

RESULTSAs compared with corresponding noncancerous tissue, there were totally 149 up-regulating genes/ESTs and 238 down-regulating genes/ESTs in gastric cancer, including 29 genes with 3-fold over-expression ratio and 21 genes with 5-fold under-expression. Fifteen protein spots were identified successfully, among whom there were ten over-expressed and five under-expressed proteins in gastric cancer tissue compared with normal tissue. Most of over-expressed genes and proteins were related to cell motility, cell proliferation, signal transduction, while those under-expressed genes and proteins were related to defense response, toxoid metabolism.

CONCLUSIONStudying gastric cancer at transcriptome and proteome levels can help demonstrate tumorigenesis and biological characteristics of gastric cancer comprehensively and provide powerful tools to find new biomarkers associated with gastric cancer and therapy targets.

Electrophoresis, Gel, Two-Dimensional ; Expressed Sequence Tags ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Genes, Neoplasm ; Genome, Human ; Humans ; Neoplasm Proteins ; Neoplasm Staging ; Oligonucleotide Array Sequence Analysis ; Proteome ; metabolism ; Proteomics ; Stomach Neoplasms ; genetics ; metabolism ; pathology

BACKGROUNDPorcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore, we investigated the liver expression profile of a highly inbred minipig line.

METHODSA cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme.

RESULTSAlignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences.

CONCLUSIONThese newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

Animals ; Expressed Sequence Tags ; Gene Library ; Liver ; metabolism ; Sequence Alignment ; Swine ; Swine, Miniature ; Transplantation, Heterologous

BACKGROUNDRetina is important in converting light into neural signals, but little is known about the regulatory genes essential for the retinal morphological formation, development and functional differentiation. This study aimed to investigate the mRNA expression patterns and cellular or subcellular distribution of 33 differentially expressed genes in the retina belonging to the early and middle-late embryogenesis stages as well as the early adult stage during human development.

METHODSIn situ hybridization and real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) were used to assay 33 differentially expressed genes which were screened out using microarray analysis and were not present in the retinal cDNA or the Expressed Sequence Tags (EST) database of the National Eye Institute (NEI) Genebank.

RESULTSNine of the 33 genes belonged to EST or the unknown cDNA fragments, and the remaining belonged to the novel genes in the retina. During the human retinal development 17 genes were down-regulated, 6 were up-regulated and the remaining 10 were relatively unchanged. Most of the genes expressed in all layers of the retina at the gestation stage, and in the fully developed retina some genes examined did show higher expression level in certain specific cells and structures such as retinal ganglion cells or the outer segment of photoreceptor cells.

CONCLUSIONThe gene expression profile during retinal development possesses temporal and spatial distribution features, which can provide experimental evidence for further research of the functions of those genes.

Expressed Sequence Tags ; Fetus ; metabolism ; Gene Expression Profiling ; Humans ; In Situ Hybridization ; RNA, Messenger ; analysis ; Retina ; embryology ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction