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

BACKGROUNDEvidence for the importance of genetic factors in male infertility is accumulating. This study was designed to identify a novel testis-specific gene related to spermatogenesis by a new strategy of digital differential display (DDD).

METHODSBased on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene. Multi-tissue RT-PCR was performed to analyse its tissue-specific expression. The full-length cDNA of the new gene was obtained using the BLAST program. Sequencing was performed and the result was analysed. Semi-quantitative RT-PCR and Northern blot analyses of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene. The sequence of the opening reading frame was integrated into the pQE-30 vector expressed in Escherichia coil strain M15 (pREP4). With IPTG induction, the target protein was detected.

RESULTSA full-length cDNA sequence of the new gene named SPATA12 (GeneBank accession number AY221117) in human testis was identified. SPATA12 was 2430 bp in length, located in chromosome 3p21.1-3p21.2. The sequence of the opening reading frame was 676-1248 bp, as was confirmed by RT-PCR and sequencing. The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20417.8 and isoelectric point of 5.23. The sequence has no significant homology with any known protein in databases. Semi-quantitative RT-PCR and Northern-blot analyses of multiple tissues showed that SPATA12 was expressed significantly in normal human testis. The expression recombinant of SPATA12 was constructed and a high level of the histidine-tagged fusion protein was obtained.

CONCLUSIONSDDD can be confirmed by SPATA12 as a novel computational biology-based approach for identification of the testis-specific expression genes. SPATA12 may function as a testicular germ cell associated gene that plays some roles in spermatogenesis. Moreover, a great amount of SPATA12 protein could be obtained by the gene recombination technique, thus providing a reliable foundation for investigating the biological function of this new protein.

Amino Acid Sequence ; Base Sequence ; Escherichia coli ; genetics ; Expressed Sequence Tags ; Gene Library ; Genes ; Humans ; Male ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatogenesis ; genetics ; Testis ; metabolism

OBJECTIVETo clonea a novel gene related to blood glucose regulation.

METHODSContrast rat model of autonomous regulation through jugular vein right atrium intubation of high concentration of glucose, the control rats were injected with 0.9% NaCl and skeletal muscle was separated. The differentially expressed fragments were identified by differential display technology (DDRT-PCR). After slot blot and Northern blot analysis, we excluded the artificial positive fragments and got the true EST (expression sequence tag) differentially expressed. These positive EST were used as probes to screen cDNA library of rat skeletal muscle, the coding region of full-length gene was cloned to pEGFP and L-6TG cell line was cultured and transiently transfected with the lipofectamine reagent. After 48 h, intact cells were examined with fluorescence microscope.

RESULTSWe got a novel full-length cDNA, named as Fang-1. GenBank Accession No. is AF399873. Using blast software (NCBI), we found Fang-1 is rat homologue of human AK001644. They share 82% identical nucleotides, indicating the family proteins are very conserved. After high concentration of glucose stimulation compared with control rats, the expression of Fang-1 was up-regulated and the expression product of Fang-1 was localized in both cytoplasm and cell nucleus.

CONCLUSIONSA novel gene related to blood glucose regulation was cloned from rat skeletal muscle. The expression product of Fang-1 was localized in both cytoplasm and cell nucleus. The gene can regulate blood glucose level by controlling some mechanisms unknown now with down-regulation expression.

Amino Acid Sequence ; Animals ; Blood Glucose ; metabolism ; Expressed Sequence Tags ; Gene Expression ; Molecular Sequence Data ; Muscle, Skeletal ; metabolism ; Rats ; Rats, Sprague-Dawley

To obtain an initial overview of gene diversity and expression pattern in porcine thymus, 11,712 ESTs (Expressed Sequence Tags) from 100-day-old porcine thymus (FTY) were sequenced and 7,071 cleaned ESTs were used for gene expression analysis. Clustered by the PHRAP program, 959 contigs and 3,074 singlets were obtained. Blast search showed that 806 contigs and 1,669 singlets (totally 5,442 ESTs) had homologues in GenBank and 1,629 ESTs were novel. According to the Gene Ontology classification, 36.99% ESTs were cataloged into the gene expression group, indicating that although the functional gene (18.78% in defense group) of thymus is expressed in a certain degree, the 100-day-old porcine thymus still exists in a developmental stage. Comparative analysis showed that the gene expression pattern of the 100-day-old porcine thymus is similar to that of the human infant thymus.
Animals ; Computational Biology ; Expressed Sequence Tags ; Fetus ; metabolism ; Gene Expression Profiling ; Genetic Variation ; Sequence Analysis, DNA ; Sus scrofa ; genetics ; metabolism ; Thymus Gland ; metabolism

This study examined the gene expression patterns of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE) by using serial analysis of gene expression (SAGE) technology. Following the construction of serial analysis of gene expression (SAGE) library of PBMCs collected from 3 cases of familial SLE patients, a large scale of tag sequencing was performed. The data extracted from sequencing files was analyzed with SAGE 2000 V 4.5 software. The top 30 expressed genes of SLE patients were uploaded to http://david.niaid.nih.gov/david/ease.htm and the functional classification of genes was obtained. The differences among those expressed gene were analyzed by Chi-square tests. The results showed that a total of 1286 unique SAGE tags were identified from 1814 individual SAGE tags. Among the 1286 unique tags, 86.8% had single copy, and only 0.2% tags had more than 20 copies. And 68.4% of the tags matched known expressed sequences, 41.1% of which matched more than one known expressed sequence. About 31.6% of the tags had no match and could represent potentially novel genes. Approximately one third of the top 30 genes were ribosomal protein, and the rest were genes related to metabolism or with unknown functions. Eight tags were found to express differentially in SAGE library of SLE patients. This study draws a profile of gene expression patterns of PBMCs in patients with SLE. Comparison of SAGE database from PBMCs between normal individuals and SLE patients will help us to better understand the pathogenesis of SLE.
Expressed Sequence Tags ; Gene Expression Profiling/*methods ; Leukocytes, Mononuclear/*metabolism ; Lupus Erythematosus, Systemic/blood ; Lupus Erythematosus, Systemic/*genetics ; Sequence Tagged Sites ; Transcription, Genetic

The mammalian liver has a very strong regeneration capacity after partial hepatectomy (PH). To further learn the genes participating in the liver regeneration (LR), 551 cDNAs selected from subtracted cDNA libraries of the regenerating rat liver were screened by microarray, and their expression profiles were studied by cluster and generalization analyses. Among them, 177 genes were identified unreported and up- or down-regulated more than twofold at one or more time points after PH, of which 62 genes were down-regulated to less than 0.5; 99 genes were up-regulated to 2-10 folds, and 16 genes were either up- or down-regulated at different time points during LR. By using BLAST and GENSCAN, these genes were located on responsible chromosomes with 131 genes on the long arms of the chromosomes. The cluster and generalization analyses showed that the gene expression profiles are similar in 2 and 4, 12 and 16, 96 and 144 h respectively after PH, suggesting that the actions of the genes expressed in the same profiles are similar, and those expressed in different profiles have less similarity. However, the types, characteristics and functions of the 177 genes remain to be further studied.
Animals ; Chromosome Mapping ; Expressed Sequence Tags ; Gene Expression Profiling ; Liver Regeneration ; genetics ; physiology ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sequence Analysis, DNA ; Time Factors

OBJECTIVETo isolate expressed sequence tags (ESTs) related to K562 cells erythroid differentiation.

METHODSModified differential display reverse transcription polymerase chain reaction (DDRT-PCR) method was applied to identify differential ESTs in uninduced and induced K562 cells by HEMIN for 36 hours. Remarkable differential ESTs were firstly selected for cloning, sequencing and bioinformational analyzing. Several ESTs representing new sequence or providing functional clue were selected for Northern blot analysis.

RESULTSSixty differentially expressed cDNA fragments related to K562 cells inducted into erythroid differentiation by HEMIN were obtained. Among them, 38 were upregulated and 22 downregulated. Among the 40 differential ESTs selected for cloning, sequencing and bioinformationally analyzing, 23 were found to match to known GenBank sequences and 10 represented cDNA sequences with only dbEST database matches and 7 ESTs have no any database matches. The results of 6 in 8 ESTs selected for Northern blot analysis were shown to be consistent with the differential expressions of DDRT-PCR.

CONCLUSIONSThe improved DDRT-PCR method had successfully overcome the problem of false positive. These ESTs provide some clue for studying the molecular mechanisms and regulation network of erythroid differentiation.

Cell Differentiation ; drug effects ; Cell Transformation, Neoplastic ; drug effects ; Erythroid Cells ; cytology ; Expressed Sequence Tags ; Hemin ; pharmacology ; Humans ; K562 Cells ; cytology ; metabolism ; Sequence Tagged Sites

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

AIMThe process of vascular calcification involves various genetic alterations which may play a very important role in the vascular calcification. Vascular smooth muscle cells undoubtedly composed the main part of vascular cells, and are involved in vascular calcification. So bovine artery smooth muscle cell (BASMC) was used to investigate the gene changes during BASMC's calcification.

METHODSBovine artery smooth muscle cells cultured in vitro was induced calcified by beta-Glycerophosphate (beta-GP). Using DD-PCR technique to screening differentially expressed genes and those differentially expressed bands were reexamined by reverse Northern blot. All the ESTs were sequenced and BLAST with GenBank.

RESULTSTotal 65 cDNAs were isolated as differentially expressed genes and 40 of them were successfully reamplified. Using reverse-Northern blot, seven of these 40 cDNAs were reproducibly expressed differentially between the two cells. Three of them are new bands and have not been reported before.

CONCLUSIONThis is the first time using DD-PCR to screen differentially expressed genes of BASMC calcification. Seven related ESTs were identified relating to BASMC calcification.

Animals ; Arteriosclerosis ; genetics ; metabolism ; pathology ; Cattle ; Cells, Cultured ; Expressed Sequence Tags ; Genetic Variation ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; pathology ; Vascular Calcification ; genetics ; metabolism ; pathology

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