A double-stranded RNA (dsRNA) mycovirus was detected in malformed fruiting bodies of Pleurotus ostreatus strain ASI2792, one of bottle cultivated commercial strains of the edible oyster mushroom. The partial RNA-dependent RNA polymerase (RdRp) gene of the P. ostreatus ASI2792 mycovirus (PoV-ASI2792) was cloned, and a cDNA sequences alignment revealed that the sequence was identical to the RdRp gene of a known PoSV found in the P. ostreatus strain. To investigate the symptoms of PoV-ASI2792 infection by comparing the isogenic virus-free P. ostreatus strains with a virus-infected strain, isogenic virus-cured P. ostreatus strains were obtained by the mycelial fragmentation method for virus curing. The absence of virus was verified with gel electrophoresis after dsRNA-specific virus purification and Northern blot analysis using a partial RdRp cDNA of PoV-ASI2792. The growth rate and mycelial dry weight of virus-infected P. ostreatus strain with PoV-ASI2792 mycovirus were compared to those of three virus-free isogenic strains on 10 different media. The virus-cured strains showed distinctly higher mycelial growth rates and dry weights on all kinds of experimental culture media, with at least a 2.2-fold higher mycelial growth rate on mushroom complete media (MCM) and Hamada media, and a 2.7-fold higher mycelial dry weight on MCM and yeastmalt-glucose agar media than those of the virus-infected strain. These results suggest that the infection of PoV mycovirus has a deleterious effect on the vegetative growth of P. ostreatus.
Agar ; Agaricales* ; Blotting, Northern ; Clone Cells ; Culture Media ; DNA, Complementary ; Electrophoresis ; Fruit ; Fungal Viruses* ; Methods ; Pleurotus* ; RNA Replicase ; RNA, Double-Stranded ; Weights and Measures

Our previous study revealed that spaceflight induced biological changes in human cervical carcinoma Caski cells. Here, we report that 48A9 cells, which were subcloned from Caski cells, experienced significant growth suppression and exhibited low tumorigenic ability after spaceflight. To further understand the potential mechanism at the transcriptional level, we compared gene expression between 48A9 cells and ground control Caski cells with suppression subtractive hybridization (SSH) and reverse Northern blotting methods, and analyzed the relative gene network and molecular functions with the Ingenuity Pathways Analysis (IPA) program. We found 5 genes, SUB1, SGEF, MALAT-1, MYL6, and MT-CO2, to be up-regulated and identified 3 new cDNAs, termed B4, B5, and C4, in 48A9 cells. In addition, we also identified the two most significant gene networks to indicate the function of these genes using the IPA program. To our knowledge, our results show for the first time that spaceflight can reduce the growth of tumor cells, and we also provide a new model for oncogenesis study.
Blotting, Northern ; methods ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Library ; Gene Regulatory Networks ; Humans ; Nucleic Acid Hybridization ; methods ; Space Flight ; Up-Regulation ; Uterine Cervical Neoplasms ; genetics ; pathology

OBJECTIVETo establish a convenient realtime PCR which can detect microRNAs in the human hepatoma cell line, Huh7 cells.

METHODSTotal RNAs in Huh7 cells were extracted. MicroRNA 122, 24 and 146a were assayed by microRNA array, and then verified by Northern blot. Stem-loop RT-PCR and poly(A)-tailed RT-PCR were used to detect the above microRNAs. Data were analyzed with Quantity One software and 7500 system software.

RESULTSMicroarray signal intensity of microRNA 122, 24 and 146a in Huh7 cells was 2201.49, 410.20 and 4.70, whose relative expression was confirmed as 0.0383, 0.0249, 0.0001 through Northern blot. While the poly(A)-tailed RT-PCR might only measure microRNA 122, Stem-loop RT-PCR could detect microRNA 122, 24 and 146a, whose average dCt was 2.5, 5.8 and 12.1 in accordance with microRNA array and Northern blot.

CONCLUSIONStem-loop RT-PCR can specifically and sensitively quantity microRNA levels, regardless of their abundance.

Base Sequence ; Blotting, Northern ; Carcinoma, Hepatocellular ; genetics ; metabolism ; Cell Line, Tumor ; DNA Primers ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; metabolism ; MicroRNAs ; analysis ; genetics ; metabolism ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo screen and identify differentially expressed genes between a fertile patient and another infertile patient who belonged to a large Chinese pedigree affected with androgen insensitivity syndrome (AIS).

METHODSWe constructed the forward and reversed subtracted libraries using genital skin fibroblasts (GSF), which were obtained from the fertile patient MJ and infertile patient ZGJ, as tester respectively. Candidate clones were screened with colony in situ hybridization, dot blot, and Southern blot analysis step by step and conformed with Northern blot analysis. The potential positive clones were sequenced and the homology of the sequences was analyzed.

RESULTSThe forward and reversed subtracted libraries containing differentially expressed pattern of two GSF cell lines were constructed. Two positive clones identified by Northern blot were obtained in the reversed subtracted library. Eleven candidate clones from the two libraries that failed to hybridize with both RNA populations were obtained simultaneously, which might represent differentially expressed low abundance transcripts. Sequencing results and homology analysis demonstrated that the two positive clones were significantly homologous with the genes of autotaxin-t and calcium binding protein calcyclin (S100A6), respectively.

CONCLUSIONSTwo positive clones and eleven clones showing no hybridization signals may represent differentially expressed genes between the two GSFs. This finding may be useful to elucidate the molecular mechanisms leading to phenotypic variation and preserved fertility of the AIS pedigree.

Androgen-Insensitivity Syndrome ; complications ; genetics ; Blotting, Northern ; Fertility ; genetics ; Fibroblasts ; cytology ; Gene Expression Profiling ; Gene Library ; Genitalia, Male ; cytology ; Humans ; In Vitro Techniques ; Infertility, Male ; etiology ; genetics ; Male ; Nucleic Acid Hybridization ; methods ; Pedigree ; Polymerase Chain Reaction ; Skin ; cytology

OBJECTIVETo screen and identify heterogeneous phenotype-associated genes of human bladder transitional cell carcinoma.

METHODSSubtractive cDNA libraries was established by means of suppression subtractive hybridization (SSH) on the basis of two human bladder transitional cell carcinoma cell lines (BLZ-211 and BLS-211) derived from the same patient, which had similar changes in chromosomes but different cell phenotypes (in terms of cell shape, susceptibility to 5-Fu and tumorigenic capacity). The positive clones in the library were selected for screening differentially expressed gene fragments by sequence analysis and blasting, and Northern blotting was performed to confirm the differentially expressed genes.

RESULTSThe subtractive forward and reverse cDNA libraries consisted of 168 and 206 white clones containing 200-700 bp inserts. After differential screening, 55 cDNA clones containing 35 different transcripts were obtained, among which 15 were identified by homology analysis as known genes (such as those coding for vimentin, keratin 7, dihydrodiol dehydrogenase and thioredoxin reductase), 11 as unknown genes, and 9 as new ESTs (GenBank dbEST database accession number DY505708, DY230447-8, DR008207).

CONCLUSIONSSH is a powerful method for identifying differentially expressed genes between different cell lines or clones, and characterization of the identified genes may provide useful information for understanding the genes responsible for different cell phenotypes.

Blotting, Northern ; Carcinoma, Transitional Cell ; genetics ; pathology ; Cell Line, Tumor ; Expressed Sequence Tags ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Library ; Genetic Heterogeneity ; Humans ; Molecular Sequence Data ; Nucleic Acid Hybridization ; methods ; Phenotype ; Sequence Analysis, DNA ; Urinary Bladder Neoplasms ; genetics ; pathology

Free-living amoebae of the genus Acanthamoeba are causative agents of granulomatous amebic encephalitis and amebic keratitis. Because the virulence of Acanthamoeba culbertsoni cultured in the laboratory is restored by consecutive brain passages, we examined the genes induced in mouse brain-passaged A. culbertsoni by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Enhanced A. culbertsoni virulence was observed during the second mouse brain passage, i.e., infected mouse mortality increased from 5% to 70%. Ten cDNAs induced during mouse brain passage were identified by DDRT-PCR and this was confirmed by northern blot analysis. BlastX searches of these cDNAs indicated the upregulations of genes encoding predictive NADH-dehydrogenase, proteasomal ATPase, and GDP-mannose pyrophosphorylase B, which have previously been reported to be associated with A. culbertsoni virulence factors.
Virulence/genetics ; Up-Regulation ; Serial Passage ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Molecular Sequence Data ; Mice, Inbred ICR ; Mice ; Genes, Protozoan/genetics ; *Gene Expression Regulation ; Gene Expression Profiling/methods ; DNA, Protozoan/biosynthesis/*physiology ; DNA, Complementary/biosynthesis ; Cloning, Molecular/methods ; Brain/parasitology ; Blotting, Northern/methods ; Animals ; Amebiasis/mortality/*parasitology ; Acanthamoeba/*genetics/*pathogenicity

OBJECTIVECloning and screening a novel candidate gene related to developing mouse cleft palate.

METHODSThe differentially expressed genes were cloned by modified PCR-based subtractive hybridization. After identification using reverse dot blotting, positive clones were sequenced and analyzed for homology in GenBank databases.

RESULTSFour novel express sequence tags were obtained, one of which spanning 809 bp was the full-length of the novel gene cDNA identified by Northern hybridization.

CONCLUSIONA novel candidate gene related to mouse cleft palate was cloned.

Adaptor Proteins, Signal Transducing ; genetics ; Amino Acid Sequence ; Animals ; Base Sequence ; Blotting, Northern ; Cleft Palate ; genetics ; pathology ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Female ; Genetic Predisposition to Disease ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; Pregnancy ; Sequence Analysis, DNA

Fluorescence differential display (FDD) technique was used to identify genes that are specifically or preferentially expressed in different developmental stages of cotton fiber cells. One hundred and nine differentially displayed cDNA fragments were isolated using 9, 21 and 27 DPA (days postanthesis) fibers as experimental materials. By a combination of two rounds of reverse Northern hybridization and Northern blot analyses, a number of such cDNA fragments were proved to represent fiber-specific/preferential genes. Sequencing determination and database searching indicated that most of these genes are novel. This work is an important step towards cloning the full-length cDNAs and characterizing the cellular functions of aforementioned genes in fiber development.
Blotting, Northern ; Cotton Fiber ; Fluorescence ; Gene Expression Profiling ; methods ; Gossypium ; genetics ; growth & development ; Polymerase Chain Reaction

OBJECTIVETo clone and screen genes related to multidrug resistance (MDR) in leukemia.

METHODSSuppression subtractive hybridization (SSH) was performed to profile differentially expressed genes between a MDR leukemia cell line (K562/DOX, as tester) and its parent cell line (K562, as driver). Reverse Northern dot blot was carried out to further screen the subtracted cDNA library. The overexpressed cDNA fragments in K562/DOX cells were sequenced and compared with known genes in Genbank. RT-PCR and Northern blot were employed to confirm the differential expression of some identified genes.

RESULTSEleven genes were identified being overexpressed in K562/DOX, including S3 ribosomal protein (S3rp) gene, NADH dehydrogenase subunit 2 (ND2) gene and My023 gene, which have not been reported to be related to MDR in cancer.

CONCLUSIONSeveral genes, which might be involved in MDR were identified, indicating novel mechanisms of MDR in leukemia.

Blotting, Northern ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Gene Library ; Genes, MDR ; genetics ; Humans ; K562 Cells ; Leukemia ; genetics ; NADH Dehydrogenase ; genetics ; Nucleic Acid Hybridization ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosomal Proteins ; genetics

OBJECTIVETo identify hemin-induced gene expression in K562 cells.

METHODSPoly A(+) RNAs were isolated from hemin-induced (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNAs were synthesized by reverse transcription. The forward subtracted cDNA library was constructed by using suppression subtractive hybridization (SSH) techniques. The recombinant plasmids were extracted and the positive clones were identified by EcoR I digestion after the amplification and screening of the library. The inserts were amplified by PCR. The upregulated cDNA transcripts were identified by reverse dot blot hybridization, DNA sequencing and homology analysis with GenBank database "blast" respectively.

RESULTSFifteen upregulated clones were identified and most of them were homologous to the mRNA sequences of protein with known function, including globin epsilon1, glutathione S-transferase like glutathione transferase Omega (GSTTLp28), selenoprotein X1 (SEPX1), triosephosphate isomerase (TPI1), ribosomal protein L7 (RPL7), ribosomal protein S13 (RPS13), ferritin light polypeptide, globin A gamma, RAD 51 homolog C(RAD51C), ferritin heavy polypeptide, X-box binding protein (XBP1). A part of the hemin-induced cDNA clones exhibited sequence similarities to that of the GenBank registered mRNA with unknown function of their expressed proteins, including the cDNA clones of DKFZp434I116, hypothetical protein HSPC014 and NOL1R2 proteins.

CONCLUSIONSHemin mainly induces the genes expression related to erythroid differentiation, protein synthesis and metabolism in K562 cell. There results provide comprehensive information useful for the differential gene expression in hemin-induced erythroid differentiation and for further function study of genes involved in hematopoiesis.

Blotting, Northern ; DNA, Complementary ; drug effects ; genetics ; Gene Expression ; drug effects ; Hemin ; pharmacology ; Humans ; K562 Cells ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction