A cDNA expression library of the tentacles of Sagartia rosea was constructed. The cDNA was cloned into eukaryotical expression plasmid pcDNA3. SMART protocol was used for cDNA library construction and bioinformatics analysis was carried out. 71 novel EST clones were obtained from 130 sequences in the library, of which there were 21 full-length clones, including cytolysin genes, flourescent protein, ubiquinol-cytochrome C reductase gene, elongation factor, ferritin gene riboflavin kinase gene, ribosomal protein. This provides a base for further investigating their biological activity and application.
Animals ; DNA, Complementary ; chemistry ; isolation & purification ; Gene Library ; RNA ; isolation & purification ; Sea Anemones ; genetics

OBJECTIVEExpress a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.

METHODSWe express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.

RESULTSThe plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15.

CONCLUSIONSkod-ssb may in future be used to enhance DNA and cDNA amplification.

Archaeal Proteins ; genetics ; isolation & purification ; metabolism ; Chromatography, Affinity ; DNA, Bacterial ; genetics ; metabolism ; DNA, Complementary ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; isolation & purification ; metabolism ; Gene Expression ; Thermococcus ; genetics ; metabolism

Stem cell growth factor (SCGF) is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFbeta, 78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain (CRD). It has been demonstrated that hSCGFbeta is strictly species-specific in regulating he-matopoiesis. This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor. Firstly, hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR. The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione S-transferase (GST) sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37 degrees C. SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form. When induced at low temperature (28 degrees C), the recombinant protein was mostly soluble. The GST-fusion recombinant protein was subsequently purified by using affinity chromatography. The clonogenic assay revealed that, unlike hSCGFbeta, hSCGF had the granulocyte/macrophage promoting activity (GPA) for murine bone marrow GM progenitor. It is concluded that, in contrast to human SCGFbeta, the intact molecular hSCGF may have no species specificity, implying that CRD domain in human SCGFbeta does not directly bind to corresponding SCGF receptor, but may have certain biological function.
Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; isolation & purification ; Hematopoiesis ; genetics ; Humans ; Species Specificity ; Stem Cell Factor ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo study viruses infecting Pinellia ternata in China.

METHODSymptom observation, DAS-ELISA and RT-PCR detection were applied.

RESULT AND CONCLUSIONDuring a survey in early spring, SMV and CMV were both commonly distributed as main viruses infecting P. ternata collected from different areas in China. But DsMV was the virus which infected P. ternate in natural condition. The infection ratio of cultivated P. ternate by SMV and CMV were 71.4% and 14.3% respectively for 21 samples collected from Ningbo, Zhejiang province; 100% and 44.4% for 18 samples from Xiaoshan, Zhejiang province; 61.9% and 33.3% for 21 samples from Hebei province; 50.0% and 41.7% for 12 samples from Anhui province; 16.7% and 16.7% for 12 samples from Sichuan province; 31.3% and none for 16 samples from Beijing. And the infection ratio of 25 wild samples from different areas of China infected by SMV and CMV were both 20.0%.

China ; Cluster Analysis ; Cucumovirus ; genetics ; isolation & purification ; DNA, Complementary ; chemistry ; genetics ; Mosaic Viruses ; classification ; genetics ; isolation & purification ; Pinellia ; virology ; Plant Diseases ; virology ; Plants, Medicinal ; virology ; Sequence Analysis, DNA

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1x10(6) ml(-1), the percentages of conidium germination and appressorium formation were (97.98+/-0.67)% and (97.88+/-0.45)%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 microg total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA (complementary DNA) library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.
DNA, Complementary ; genetics ; isolation & purification ; DNA, Fungal ; genetics ; isolation & purification ; Gene Library ; Genes, Fungal ; Magnaporthe ; genetics ; growth & development ; pathogenicity ; Oryza ; microbiology ; Plant Diseases ; microbiology ; RNA, Fungal ; genetics ; isolation & purification

Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm.
Aldose-Ketose Isomerases ; genetics ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Mevalonic Acid ; metabolism ; Multienzyme Complexes ; genetics ; Oxidoreductases ; genetics ; Phylogeny ; RNA ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Taxus ; genetics

The cDNA encoding the rabbit metallothionein-I was amplified by RT-PCR from the rabbit liver induced by cadmium and cloned into prokaryotic fusion expression vector pQE40. Then it was transformed into Escherichia coli M15. Positive expression clones were detected by colony blotting. Target protein solubility was determined by Western blotting analysis. The optimal induction condition of the level of protein expression with IPTG induction was established by SDS-PAGE electrophoresis and ImageMaster VDS software analysis. The fusion protein can be purified from lysates with Ni-NTA agarose. We found that the fusion protein with apparent molecular weight 32 KD existed in two ways: soluble and insoluble in Escherichia coli. After 1 mM IPTG induction, the level of expression of the fusion protein increased with the prolongation of induction time and reached a peak in 9 h by ImageMaster VDS software analysis, accounting for 57.4% of all the insoluble protein. The purified fusion protein was obtained by Ni-NTA affinity chromatography. This fusion protein can be used in further studies on the preparation of MT-I protein and development of protein product.
Animals ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Metallothionein ; genetics ; metabolism ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Transformation, Bacterial

OBJECTIVETo construct the cDNA subclones spanning the entire genome of dengue 2 virus NGC strain for further construction of full-length infectious viral cDNA clone.

METHODSTwo pairs of primers were designed according to the restriction endonuclease sites in the viral genome of dengue 2 virus NGC strain. After viral RNA extraction from the brain of infected new-born mice, two parts of full-length viral cDNA were amplified by long RT-PCR and cloned into the vector pCR-XL-TOPO. The partial sequence of the recombinant plasmid was determined.

RESULTS AND CONCLUSIONSequence analysis and digestion with restriction enzymes demonstrated that the two cDNA subclones were specific for dengue 2 virus NGC strain, suggesting the successful construction of the two cDNA subclones of dengue 2 virus NGC strain.

Animals ; Animals, Newborn ; Brain ; virology ; Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; DNA, Viral ; biosynthesis ; genetics ; Dengue ; virology ; Dengue Virus ; classification ; genetics ; isolation & purification ; Genome, Viral ; Mice ; RNA, Viral ; genetics ; isolation & purification ; Recombination, Genetic ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo identify and clone the gene encoding human M96 gene and study its expression spectrum in several blood cell lines.

METHODSAccording to the sequence of human EST which was highly homologous to the mouse M96 gene, primers used for library screening were synthesized, then the human adult testis and fetal brain cDNA library were screened. The gene was analyzed by making use of BLAST and CLUSTAL W, and its expression spectrum was studied by multiple-cell lines Northern blot analysis. The expression change of M961 in cell differentiation was observed by use of K562 cell line induced by hemin.

RESULTSTwo cDNA clones encoding human M96 gene were isolated, identified and named as M961, and M962. They were found to be isoforms of each other. Northern, blot showed that M961 gene was expressed highly in CEM, Hel, Dami and K562 cell lines. However, during K562 cell line differentiation, process the expression of M961 elevated only slightly.

CONCLUSIONSM961 gene was expressed highly in pluripotent cell lines with erythrocytic and megakaryocytic potentials.

Alternative Splicing ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Neoplasm ; genetics ; isolation & purification ; Hemin ; pharmacology ; Humans ; K562 Cells ; Molecular Sequence Data ; Protein Isoforms ; genetics ; isolation & purification ; Protein Splicing ; Zinc Fingers ; genetics

OBJECTIVETo explore the molecular characteristics and molecular variation of human rotavirus (HRV) strains and to understand the relationship between clinical characteristics and epidemiology of different HRV-VP7 and NSP4.

METHODSDouble-strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 were amplified with RT-PCR. Then cDNAs were sequenced and compared with 4 human rotavirus NSP4 (Wa, KUN, AU-1, Hochi)) and 3 animal rotavirus NSP4 (EW, OSU, SA11) available in the GenBank while the epidemic strains of human rotavirus isolated in different areas of China were compared, using the Clustal-mp, DNAssist, MEGA2 software. The G serotype of VP7 was analysed by PCR.

RESULTSSerotype G1 was prevalent in 2002 while serotype G3 was the prevalent in Kumming in 2003. The NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 belonged to Wa with highly conservative amino acid. Samples isolated in the same years but not in the same area shared higher homology. Symptoms associated with heavy diarrhea did not seem to be associated with NSP4 molecular variation (P > 0.05).

CONCLUSIONObvious variations of VP7 typing were seen in the same season, as well as in different areas and years. Due to the stable nature of NSP4, it seem to be a better candidate for vaccine production, than VP7.

China ; DNA, Complementary ; genetics ; DNA, Viral ; Genes, Viral ; Humans ; Polymerase Chain Reaction ; RNA, Double-Stranded ; genetics ; RNA, Viral ; RNA-Directed DNA Polymerase ; Rotavirus ; classification ; genetics ; isolation & purification ; Rotavirus Vaccines ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Serotyping