Differential cDNA Fragments Research Articles

Differential Gene Expression for Curvularia eragrostidis Pathogenic Incidence in Crabgrass (Digitaria sanguinalis) Revealed by cDNA-AFLP Analysis

Gene expression profiles of Digitaria sanguinalis infected by Curvularia eragrostidis strain QZ-2000 at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP). Inoculum strength was more determinant of gene expression than dew duration. A total of 256 primer combinations were used for selective amplification and 1214 transcript-derived fragments (TDFs) were selected for their differential expression. Of these, 518 up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to be cloned and 35 of them were successfully cloned and sequenced, of which 25 were homologous to genes of known function according to the GenBank database. Only 6 genes were up-regulated in Curvularia eragrostidis-inoculated D. sanguinalis, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000 on D. sanguinalis. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control D. sanguinalis.

Identification of differentially expressed genes in Sulfobacillus sp. TPY grown on either elemental sulphur or Fe2+

Sulfobacillus sp. TPY is a moderately thermophilic and acidophilic bacterium found in hydrothermal vents in the Pacific Ocean. This bacterium can oxidize ferrous sulfate (Fe(2+)) and elemental sulfur (S(0)) under separate conditions. We used random arbitrarily primed polymerase chain reaction (RAP-PCR) to screen and identify differentially expressed genes from bacteria grown on Fe(2+) or S(0) as the energy source. Fifty-five differential cDNA fragments were isolated and subjected to single-pass sequencing. Thirty-five fragments were identified as orthologs of known genes in the GenBank databases, of which 19 were confirmed to be differentially expressed at the transcriptional level by Northern blot analysis. Among these 19 genes, 14 genes, including isocitrate dehydrogenase, formyltetrahydrofolate deformylase, 3-hydroxybutyryl-CoA dehydrogenase, and GTP-binding protein, were upregulated in TPY grown on Fe(2+) or downregulated in TPY grown on S(0), while five genes such as the outer membrane adhesion-like protein, phosphomannomutase, and cysteine desulfurase sufS were upregulated in TPY strain grown on S(0) or downregulated in TPY grown on Fe(2+). These altered genes are involved in metabolism, osmotic stress, cell membrane alterations, oxidative stress, and the regulatory adaptive response. These results will aid our understanding of the molecular basis of Fe(2+) or S(0) oxidation by the moderately thermophilic and acidophilic bacteria.

Differential expression of salt tolerance related genes in Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee

We examined salt tolerance responsive genes in Pak-choi under salt stress and analyze their potential function. The mRNA differential display was used to screen the transcript derived fragments (TDFs) related to salinity tolerance in tolerant and moderately tolerant Pak-choi germplasm. Seventy-eight primer combinations generated 101 differential cDNA fragments, which were divided into 10 expression types. Seven cDNA sequences (GenBank accession Nos. DQ006915~DQ006921) obtained and sequenced were highly homologous to some known expression genes or the genes related to the signaling pathways in plants under different abiotic stress.

Identification of Differentially Expressed Genes from Rhodothermus sp. XMH10 in Response to Low Temperature Using Random Arbitrarily Primed PCR

Most research on the adaptation of thermophiles is focused on their adaptation to heat stress; only a few studies are focused on their cold adaptation. In this report, the thermophilic bacterium Rhodothermus sp. XMH10 was examined to gain a better understanding of gene expression in response to low temperature. Random arbitrarily primed polymerase chain reaction (RAP-PCR) was used to isolate and identify differentially expressed genes of bacteria grown at 45 degrees C (lowest) compared to those at 75 degrees C (optimal). Fifty-three differential cDNA fragments in total were isolated. Among them, 35 different cDNAs were analyzed by Northern blot, and 17 were confirmed to be differentially expressed at the transcriptional levels. These genes reflected a profile of differential expression and were involved in many physiological processes such as metabolism, cell membrane alterations, and regulatory adaptive response; most of them have never been previously reported. This study provides some new information on the adaptation of thermophilic bacteria to environmental temperature stress.

Cloning of Salt Stress Responsive cDNA from Wheat and Resistant Analysis of Differential Fragment SR07 in Transgenic Tobacco

Analysis of the gene expression differentiation in leaves of wheat ( Triticum aestivum L.) cultivar Baofeng 7228, under salt stress, was carried out by Differential-Display Reverse Transcription-polymerase Chain Reaction (DDRT-PCR.) Twenty-seven differential cDNA fragments were obtained. The expression of the SR07 fragment was induced noticeably by salt treatment, and the nucleotide sequence homology of 87% between the SR07 fragment and PIPs (water channel proteins) was observed. Further research showed that a 561 bp open read frame was present in the SR07 fragment. Plant expression vector of pCAMBIA-SR07 was constructed and three transformants of tobacco ( Nicotiana tobacum) mediated by Agrobacterium tumefaciens plasmid were obtained. Resistance to salt, PEG, and mannitol stresses of the three transformants were examined. No significant difference ( P > 0.05) was observed between the control and the transformants in resistance to salt stress, but there was significant difference ( P < 0.05) between the control and the transformants in resistance to PEG and mannitol stresses. Therefore, the expression of the SR07 fragment may play an important role in the water regulation of the plant.

Circadian expression of clock and screening of clock-controlled genes in peripheral lymphocytes of rat

In this paper, the circadian pattern of Clock and genes mediated by the Clock was investigated in peripheral lymphocytes of rats. Circadian rhythms of Clock are found under the regimes of constant darkness (DD) and 12-h light–12-h dark (LD12:12 h), with the peak phase at CT7 and ZT21, respectively. Ten differential cDNA fragments were identified to be mediated by the Clock, including three known genes ( catalase, myelin proteolipid protein, and histone acetylase), four known expressed sequence tags (ESTs), and three novel ESTs. Experiment of the RNA interference revealed that these ESTs were down-regulated by the Clock gene and three of them were identified as clock-controlled genes. Understanding of clock-mediated genes may lead to a new direction in drug design for control of circadian rhythms.

Identification of antiviral-relevant genes in the cultured fish cells induced by UV-inactivated virus

UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (crucian carp (Carassius auratus L.) blastulae) cells, and thus defend host cells against the virus invasion. The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antiviral-relevant genes. In this study, suppressive subtractive hybridization is applied to constructing a subtracted cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells. 272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library. Sequencing analysis reveals 69 genes, including 46 known gene homologues, and 23 unknown putative genes. The known genes include the genes involved in interferon signaling pathways, such as Stat1 and Jak1, the antiviral genes, such as Mx and Viperin, and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknown putative genes contain AU-rich element in their sequences. Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR. The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells, but also leads to the expression of a series of antiviral-relevant genes or immune-relevant genes, and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.

Genes responding to vernalization in hexaploid wheat.

Genotype-specific gene expression in response to vernalization in common wheat was examined by the differential display method. Two near-isogenic lines of Vrn-A1 ( Vrn-A1 for the spring type and vrn-A1 for the winter type) were treated by vernalization of developing embryos in detached-ear cultures. This treatment was effective to promote vrn-A1 genotypes to head at a time equivalent to that of Vrn-A1. Differential cDNA fragments were isolated by the RT-PCR method from embryos subjected to vernalization treatments for 2- and 4-weeks at DAP10 and DAP20 stages, respectively. Among 110 differential cDNA fragments isolated, 48 were examined for their chromosomal locations and designated as wec ( wheat- embryo cold treatment) genes. Seven wec genes showed genotype-specific expression in response to vernalization. The statistical analysis utilizing two recombinant inbred lines showed that four wec genes were significantly associated with heading factors.

Direct effect of croton oil on intestinal epithelial cells and colonic smooth muscle cells

To investigate the direct effect of croton oil (CO) on human intestinal epithelial cell (HIEC) and guinea pig colonic smooth muscle cells in vitro. Growth curves of HIEC were drawn by MTT colorimetry. The dynamics of cell proliferation was analyzed with flow cytometry, and morphological changes were observed under light and electron microscopy after long-term (6 weeks) treatment with CO. Expression of cyclooxygenase-2 (COX-2) mRNA was detected by dot blot in HIEC treated with CO. Genes related to CO were screened by DD-PCR, and the direct effect of CO on the contractility of isolated guinea pig colonic smooth muscle cells was observed. High concentration (20-40 mg x L(-1)) CO inhibited cell growth significantly (1, 3, 5, 7d OD sequence: (20 mg x L(-1)) 0.040+/-0.003, 0.081+/-0.012, 0.147+/-0.022,0.024+/-0.016; (40 mg x L(-1)) 0.033+/-0.044, 0.056+/-0.012, 0.104+/-0.010, 0.189+/-0.006; OD control 0.031+/-0.008, 0.096+/-0.012, 0.173+/-0.009, 0.300+/-0.016, P<0.01), which appeared to be related directly to the dosage. Compared with the control, the fraction number of cells in G1 phase decreased from 0.60 to 0.58, while that in S phase increased from 0.30 to 0.34 and DNA index also increased after 6 weeks of treatment with CO (the dosage was increased gradually from 4 to 40 mg x L(-1)). Light microscopic observation revealed that cells had karyomegaly, less plasma and karyoplasm lopsidedness. Electron microscopy also showed an increase in cell proliferation and in the quantity of abnormal nuclei with pathologic mitosis. Expression of COX-2 mRNA decreased significantly in HIEC treated with CO. Thirteen differential cDNA fragments were cloned from HIEC treated with CO, one of which was 100 percent homologous with human mitochondrial cytochrome C oxidase subunit II. The length of isolated guinea pig colonic smooth muscle cells was significantly shortened after treatment with CO (P<0.05). At a high CO concentration (>20 mg x L(-1)), cell growth and proliferation are inhibited in a dosage-dependent manner. Increase in cell proliferation and in malignant conversion of the cellular phenotype is observed in cells cultured chronically with CO. COX-2 mRNA expression decreases significantly, while human mitochondrial cytochrome C oxidase subunit IImRNA expression increases significantly in HIEC treated with CO. CO also has a direct effect on the contractility of Guinea pig colonic smooth muscle cells.

Isolation of cDNA fragment of fertility-related gene in photoperiod-sensitive genic male sterile rice

The photoperiod-sensitive genic male sterile rice (PGMR) is particularly useful to take advantage of heterosis in rice. mRNA differential display was used to isolate the fertility-relative genes in rice. After establishing an optimized mRNA differential display system, one of the differential cDNA fragments that maybe related to the development and maturation of rice panicle was cloned from a PGMR Nongken 58s.

MRNA differential display between sterile and fertile anther of rice and analysis of cDNA differential fragments

Rice anther and leaf mRNAs from two cytoplasmic male sterile lines (Maxie A and Congguong 41A) were compared with those from their maintainers and F(1) hybrids by using mRNA differential display to study gene expression pattern in sterile anther during pollen abortion. Anther cDNA differential bands between sterile and fertile plants were more than those of leaf. It was indicated that the expression of fertility gene(s) in the anther was more activable and sufficient than in the leaf. The gene transcript pattern in one of different types of anther was not only associated with its pollen sterility degree but also with its stage of pollen abortion. The anthers with full or partial sterility or with early abortion produced more cDNA differential bands than those fertile or late abortion anthers. In twelve recovered differential cDNA fragments, two were probably associated with male sterility, i. e. one is (AB(4)A(5)), which was specifically expressed in the sterile anther, and the other (AB(3)B(2)), which contained some sequence homologous to a mitochondrial gene (coxII) and whose expression was partially suppressed in the sterile anther.

Suppression in transformed avian fibroblasts of a gene (CO6) encoding a membrane protein related to mammalian potassium channel regulatory subunits.

Gene expression patterns in normal and v-myc-transformed quail embryo fibroblasts were compared by mRNA differential display. Displaying approximately 2500 mRNA species by reverse transcription/PCR, reamplification of 73 differential cDNA fragments and rescreening by Northern analysis led to the isolation of a clone, termed CO6, that hybridized to an mRNA species present only in the normal but not in the transformed fibroblasts. Further analyses revealed that the 0.95-kb CO6 mRNA was present in all normal quail and chicken embryo fibroblasts tested, but that it was undetectable in a variety of established quail cell lines transformed by the v-myc, v-myc/v-mil, v-jun/junD or v-src oncogenes or by a chemical carcinogen. Furthermore, CO6 mRNA was not detectable in fibroblasts newly transformed by retroviral constructs carrying v-myc or v-jun alleles or by the avian sarcoma virus ASV17. In fibroblasts transformed by a temperature-sensitive v-src mutant, expression of CO6 was strongly induced at the non-permissive temperature and reduced at the permissive temperature. Nucleotide sequence analysis of quail CO6 cDNA indicated that the corresponding gene encodes a 200-amino acid protein with 46 to 48% amino acid sequence identity to the regulatory beta subunits (K(VCa)beta) of the bovine, human and canine high conductance Ca2+-activated K+ channels. No sequence homology to other ion channel subunits or to any other proteins in the databases was found. Like the K(VCa)beta subunits, the CO6 protein contains two putative transmembrane segments. Based on the relationship to mammalian K(VCa)beta both in primary structure and domain topology, the CO6 protein may represent the regulatory subunit of a yet unidentified avian Ca2+-activated potassium channel or a related membrane protein possibly involved in the regulation of cell proliferation.