Telomeres are the structures that locate at the terminals of linear eukaryotic chromosomes. They can play essential roles in many cellular processes. The terminal location ofArabidopsis-type TTTAGGG tandem repeats wer...Telomeres are the structures that locate at the terminals of linear eukaryotic chromosomes. They can play essential roles in many cellular processes. The terminal location ofArabidopsis-type TTTAGGG tandem repeats were thought to be highly conserved. The terminal location of Ginkgo biloba L. consisting of TTTAGGG tandem repeats, were confirmed by Bal31 exonuclease degradation and Southern blotting. By comparing telomeric restriction fragment (TRF) lengths at different developmental stages from embryos to seedlings, a fluctuant tendency towards variation was found in these samples. The TRF length of embryos was also compared with that of embryonal calli and an upward trend was discovered in callus culture. The results suggest that there should be a telomerase mechanism or/and ALT mechanism for the maintenance of telomere length.展开更多
Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection metho...Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g<sup>-1</sup>) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.展开更多
A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93...A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93.9 mg carvacrol/kg DM. After the experimental period of 5 weeks, 20 piglets of both extreme feeding groups were slaughtered: 10 animals of the control group and 10 animals of the group that received 8 g oregano/kg. Ingesta samples of jejunum, caecum and colon were collected and analyzed by FISH and PCR RFLP to compare the diversity of microbiota. The results showed no significant changes in microbiota in response to oregano. The patterns of the PCR-RFLP showed a similarity of 61.8% - 91.8% in both feeding groups. In conclusion, an effect of oregano on the in- testinal microbiota could not be shown under the methods used.展开更多
Objectives Tumor necrosis factor-α (TNF-α) may play an important role in host's immune response to mycobacterium tuberculosis (M. tuberculosis) infection. This study was to investigate the association of TNF-α...Objectives Tumor necrosis factor-α (TNF-α) may play an important role in host's immune response to mycobacterium tuberculosis (M. tuberculosis) infection. This study was to investigate the association of TNF-α gene polymorphism with pulmonary tuberculosis (TB) among patients with coal worker's pneumoconiosis (CWP). Methods A case-control study was conducted in 113 patients with confirmed CWP complicated with pulmonary TB and 113 non-TB controls with CWP. They were matched in gender, age, job, and stage of pneumoconiosis. All participants were interviewed with questionnaires and their blood specimens were collected for genetic determination with informed consent. The TNF-α gene polymorphism was determined with polymerase chain reaction of restriction fragment length polymorphism (PCR-RFLP). Frequency of genotypes was assessed for Hardy-Weinberg equilibrium by chi-square test or Fisher's exact probability. Factors influencing the association of individual susceptibility with pulmonary TB were evaluated with logistic regression analysis. Gene-environment interaction was evaluated by a multiplieative model with combined OR. All data were analyzed using SAS version 8.2 software. Results No significant difference in frequency of the TNF-α-308 genotype was found between CWP complicated with pulmonary TB and non-TB controls (2,2=5.44, P=-0.07). But difference in frequency of the TNF-α-308 A allele was identified between them (2,2-5.14, P=0.02). No significant difference in frequencies of the TNF-α-238 genotype and allele (P=0.23 and P=0.09, respectively) was found between cases and controls either, with combined (GG and AA) OR of 3.96 (95% confidence interval of 1.30-12.09) at the -308 locus of the TNF-α gene, as compared to combination of the TNF-α-238 GG and TNF-α-308 GG genotypes. Multivariate-adjusted odds ratio of the TNF-α-238 GG and TNF-α-308 GA genotypes was 1.98 (95% CI of 1.06-3.71) for risk for pulmonary TB in patients with CWP. There was a synergic interaction between the TNF-a-308 GG genotype and body mass index (OR=4.92), as well as an interaction between the TNF-α-308 GG genotype and history of BCG immunization or history of TB exposure. And, the interaction of the TNF-α-238 GG genotype and history of BCG immunization or TB exposure with risk for pulmonary TB in them was also indicated. Conclusions TNF-α-308 A allele is associated with an elevated risk for pulmonary TB, whereas TNF-α-238 A allele was otherwise.展开更多
A rice population consisting of 90 TN1/Guiyigu F3 lines was employed to analyze the linkage between DNA markers and a new gene Wbph6(t) conferring resistance to whitebacked planthopper, Sogatella furcifera By using th...A rice population consisting of 90 TN1/Guiyigu F3 lines was employed to analyze the linkage between DNA markers and a new gene Wbph6(t) conferring resistance to whitebacked planthopper, Sogatella furcifera By using the mapping approach of bulked extremes and recessive class, Wbph6(t) was mapped onto the short arm of chromosome 11 with a genetic distance of 21.2 cM to SSLP marker RM167.展开更多
To characterize the effects of pentachlorophenol (PCP) on the performance and microbial community of aerobic granular sludge in sequencing batch reactor (SBR), the web-based terminal restriction fragment length polymo...To characterize the effects of pentachlorophenol (PCP) on the performance and microbial community of aerobic granular sludge in sequencing batch reactor (SBR), the web-based terminal restriction fragment length polymorphism (T-RFLP) and real-time PCR (RT- PCR) techniques were used to explore the bacterial community structure. When PCP increased from 0 to 50 mg/L, the COD removal rate changed little, while the ammonia removal rate dropped from 100% to 64.9%. The results of molecular characterization showed t...展开更多
The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphi...The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCBI for identification purposes. The 90.6% of the clones were a?l...展开更多
Objective To compare the bacterioplankton communities in streams exposed to pollution of different types. Methods The bacterioplankton communities in three selected heavily polluted streams were investigated by using ...Objective To compare the bacterioplankton communities in streams exposed to pollution of different types. Methods The bacterioplankton communities in three selected heavily polluted streams were investigated by using terminal‐restriction fragment length polymorphism (T‐RFLP) analysis in combination with 16S rRNA gene clone library analysis. Results Both T‐RFLP and 16S rRNA gene clone library revealed a great difference in bacterioplankton community composition in the different streams. Conclusion This work might provide some new insights into bioremediation of heavily polluted streams.展开更多
γ -actin (ACTG1) gene is a cytoplasmic nonmuscle actin gene, which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea. Mutations in ACTG1 were found to cause autosomal dominant, progressi...γ -actin (ACTG1) gene is a cytoplasmic nonmuscle actin gene, which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea. Mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families, respectively. In this study, a novel missense mutation (c.364A〉G; p.I122V) co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls. The alteration of residue Ile122 was predicted to damage its interaction with actin-binding proteins, which may cause disruption of hair cell organization and function. These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum of ACTG1 mutations causing hearing loss.展开更多
Symbiotic algae (Symbiodinium sp.) in scleractinian corals are important in understanding how coral reefs will respond to global climate change. The present paper reports on the diversity of Symbiodinium sp. in 48 s...Symbiotic algae (Symbiodinium sp.) in scleractinian corals are important in understanding how coral reefs will respond to global climate change. The present paper reports on the diversity of Symbiodinium sp. in 48 scleractinian coral species from 25 genera and 10 families sampled from the Xisha Islands in the South China Sea, which were identified with the use of restriction fragment length polymorphism (RFLP) of the nuclear ribosomal DNA large subunit gene (rDNA). The results showed that: (i) Symbiodinium Clade C was the dominant zooxanthellae in scleractinian corals in the Xisha Islands; (ii) Symbiodinium Clade D was found in the corals Montipora aequituberculata, Galaxea fascicularis, and Plerogyra sinuosa; and (iii) both Symbiodinium Clades C and D were found simultaneously in Montipora digitata, Psammocora contigua, and Galaxeafascicularis. A poor capacity for symbiosis polymorphism, as uncovered by RFLP, in the Xisha Islands indicates that the scleractinian corals have low adaptability to environmental changes. Further studies are needed to investigate zooxanthellae diversity using other molecular markers.展开更多
A novel phytoplasma was detected in a cherry plum(Prunus cerasifera Ehrh) tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The le...A novel phytoplasma was detected in a cherry plum(Prunus cerasifera Ehrh) tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies(PLBs) were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 μm, and distributed in the phloem cells of the infected tissues. A 1 246-bp 16 S ribosomal RNA(rRNA) gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16 S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows(AY) group(16SrI group). Virtual and actual restriction fragment length polymorphism(RFLP) analysis of the 16 S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group(16SrI) by four restriction sites, Hinf I, Mse I, Sau3 A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16 S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf(CPYL) symptoms is probably a distinct and novel subgroup lineage in the AY group(16SrI). In addition, the novel phytoplasma was experimentally transmitted to periwinkle(Catharanthus roseus) plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder(Cuscuta odorata) connections.展开更多
Objective: To understand the role of mitochondrial DNA (mtDNA) in carcinogenesis. Methods: single-step method was used to isolate the mtDNA from human lung adenocarcinoma cell line SPC-A-1. The mtDNA was analyzed by r...Objective: To understand the role of mitochondrial DNA (mtDNA) in carcinogenesis. Methods: single-step method was used to isolate the mtDNA from human lung adenocarcinoma cell line SPC-A-1. The mtDNA was analyzed by restriction fragment length polymorphism (RFLP) with 11 kinds of restriction endonuclease, which were Pvu II, Xho I, Pst I, EcoR I, BstE II, Hind III, Hpa I, Bc1 I, EcoR V, Sca I and Xba I. Restriction map of mtDNA from SPC-A-1 cell was obtained by the single and double-digestion method. Results: It was found that no variation at 32 restriction-sites could be detected in the coding region of mtDNA from SPC-A-1 cell line. But a new site was found at nucleotide 16276 (EcoR V) within the noncoding region. Conclusion: These results indicate that the primary structure of gene coding region of mtDNA isolated from SPC-A-1 cell is highly stable. While the major variation of nucleotide is probably located in the noncoding region.展开更多
After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the ...After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the effect of excessive CdCl 2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl 2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl 2 did not inhibit the replication and amp + genes expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl 2 treatment for 24 hours might cause the sequences change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl 2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl 2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.展开更多
DNA from 36 patients with chronic myelogenous leukemia (CML) at various clinical stages and 6 cases of acute leukemia was investigated for alterations of the p53 gene by Southern blot analysis.Rearrangements of the p5...DNA from 36 patients with chronic myelogenous leukemia (CML) at various clinical stages and 6 cases of acute leukemia was investigated for alterations of the p53 gene by Southern blot analysis.Rearrangements of the p53 gene were seen in 3 of 12 (25.00%) cases of blast crisis and accelerated phase (AP) of CML and in only one of 18 chronic phrase (CP),just as has been reported previously. Meanwhile,by restriction fragment length polymorphism (RFLP) analysis the Bgl II site polymorphism in the p53 gene was also found. The frequency in Chinese people detected here was 0.392,which was strikingly higher than that in some other countries(P<0. 001).These results suggested that the alterations of the p53 gene, for example,p53 rearrangements,were probably responsible for the progression of BC in some CML patients, and that the frequency of Bgl II polymorphism in the p53 gene might be related to the population distribution.展开更多
Using human nov (nephroblastoma overexpressed,nov) DNA as probe,hybridization to total cellular DNAs of tumor and normal cells digested by restriction enzymes BamHI or EcoRI respectively was carried out through Southe...Using human nov (nephroblastoma overexpressed,nov) DNA as probe,hybridization to total cellular DNAs of tumor and normal cells digested by restriction enzymes BamHI or EcoRI respectively was carried out through Southern blot. It was observed that nov gene in these cells is not only highly conserved, but also certains RFLP characteristic . The correlation between RFLP characteristic of nov gene and its function was analyzed .展开更多
The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for indus...The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g<sup>-1</sup>, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g<sup>-1</sup>. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g<sup>-1</sup>. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g<sup>-1</sup>) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g<sup>-1</sup>), and the other Firmicutes (3.7 × 106 MPN g<sup>-1</sup>). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g<sup>-1</sup>), Burkhokderia sp. (1.5 × 106 MPN g<sup>-1</sup>), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g<sup>-1</sup>). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.展开更多
Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and ...Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 10<sup>6</sup> MPN g<sup>-1</sup> dry soil), followed by γ-Proteobacteria (1.32 × 10<sup>6</sup> MPN), δ-Proteobacteria (0.006 × 10<sup>6</sup> MPN), and the other gram negative bacteria (0.006 × 10<sup>6</sup> MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 10<sup>6</sup> MPN), followed by α-Proteobacteria (26.0 × 10<sup>6</sup> MPN), β-Proteobacteria (17.1 × 10<sup>6</sup> MPN), δ-Proteobacteria (11.2 × 10<sup>6</sup> MPN), the other Firmicutes (1.71 × 10<sup>6</sup> MPN), γ-Proteobacteria (0.5 × 10<sup>6</sup> MPN), and the other gram negative bacteria (0.05 × 10<sup>6</sup> MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 10<sup>6</sup> MPN), followed by Actinobacteria (2.14 × 10<sup>6</sup> MPN), Bacillus sp. (2.14 × 10<sup>6</sup> MPN), and γ-Proteobacteria (0.35 × 10<sup>6</sup> MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candidate of a new evaluation system of soil and manure.展开更多
In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial group...In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L<sup>-1</sup><sup></sup>) were included in row cattle feces (1.78 × 10<sup>4</sup> MPN g<sup>-1</sup>) and cattle feces manure (>3.84 × 10<sup>4</sup> MPN g<sup>-1</sup>). Compost originated from leftover food (>44.8 × 10<sup>4</sup> MPN g<sup>-1</sup>) and shochu lee (>320 × 10<sup>4</sup> MPN g<sup>-1</sup>) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L<sup>-1</sup><sup></sup>), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 10<sup>4</sup> MPN g<sup>-1</sup>), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L<sup>-1 </sup>of<sup> </sup>ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 10<sup>4</sup> MPN g<sup>-1</sup>), followed by cattle feces manure (4.19 × 10<sup>4</sup> MPN g<sup>-1</sup>), and shochu lee (0.36 × 10<sup>4</sup> MPN g<sup>-1</sup>), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.展开更多
Objective To establish the specific 16S-23S rRNA gene spacer regions in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis. M...Objective To establish the specific 16S-23S rRNA gene spacer regions in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis. Methods A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA from sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 26 species was amplified by PCR, and further analyzed by RFLP, DNA cloning and sequences analysis. Furthermore, all specimens were examined by bacterial culturing and PCR-RFLP analysis. The evaluation of these assays in practical clinic practice was also discussed.Results Restriction enzyme analysis revealed one, two or three bands or more observed among the 26 different standard strains. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction with human genomic DNA, fungus or virus. Fourteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf Ⅰ or Alu Ⅰ digestion. The only difference between K.pneumoniae and E.durans was located at the site of the 779th nucleotide according to the sequence analysis and only XmaⅢ digestion could distinguish one from another. Of 42 specimens from septicemic neonates, 15 were identified as positive by blood culture at a rate of 35.7%. However, 27 specimens identified as positive by PCR, with a rate of 64.2%, a method significantly more effective than blood culture (P<0.01). Of 6 cerebrospinal fluid (CSF) specimens, one tested positive for S.epidermidis was also positive by PCR, two culture negative were positive by PCR and diagnosed as S.epidermidis according to the DNA pattern. One positive for C.neoformans was negative by PCR. The other two specimens were negative by both PCR and culture.Conclusions The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in providing a new technique for detecting pathogens in clinical bacterial infections.展开更多
文摘Telomeres are the structures that locate at the terminals of linear eukaryotic chromosomes. They can play essential roles in many cellular processes. The terminal location ofArabidopsis-type TTTAGGG tandem repeats were thought to be highly conserved. The terminal location of Ginkgo biloba L. consisting of TTTAGGG tandem repeats, were confirmed by Bal31 exonuclease degradation and Southern blotting. By comparing telomeric restriction fragment (TRF) lengths at different developmental stages from embryos to seedlings, a fluctuant tendency towards variation was found in these samples. The TRF length of embryos was also compared with that of embryonal calli and an upward trend was discovered in callus culture. The results suggest that there should be a telomerase mechanism or/and ALT mechanism for the maintenance of telomere length.
文摘Lactic acid bacteria have not only been used to produce various kinds of fermented food, but also used as probiotic products. As lactic acid bacterial group was consisted from diverse genera, a simple inspection method by which numbers and contained microorganisms could be automatically analyzed without any preliminary information was required to use them more effectively. In this manuscript, lactic acid bacterial groups in commercial products of kimuchi, komekouji-miso, and yoghurt were identified and enumerated by our newly developed method [1]-[3], to evaluate whether the method could be used as an inspection method of various food samples. In kimuchi, numerically dominant bacteria were Lactobacillus sakei, and L. casei (1.4 × 104 MPN g<sup>-1</sup>) and Leuconostoc spp. (l.4 × 104 MPN). In kouji-miso, numerically dominant bacteria was Bacillus spp. (3 × 103 MPN), which mainly included B. subtilis group and B. cereus group. Lactic acid bacteria such as Lactobacillus spp., or Lactococcus spp., included in the komekouji-miso, could be enumerated after 3 days incubation (1.24 × 104 MPN), but not detected after 7 days incubation. In yoghurt A and C, Lactococcus lactis was detected as numerically dominant lactic acid bacteria (3.0 × 105 MPN). In yoghurt B, Lactobacillus spp., or Lactococcus spp., was detected not only by a culturebased method but also by an unculture-based method, although there was a difference between the both estimated numbers. The present results suggested that the method might become useful as a simple inspection method of food microorganisms, because time and labor of the analysis could be reduced by using an unculture-based method and MCE-202 MultiNA. In this study, Bifidobacteriium spp. was not detected in B and C yoghurt, in spite of indicating their existence, and numbers of lactic acid bacteria were lower than the level of the daily product regulation, because 16S rDNA of Bifidobacteriium spp. might not be amplified by the used PCR condition. The PCR condition must be changed so as to amplify Bifidobacterium spp., before the method will be used as an inspection method for lactic acid bacteria.
文摘A total of 80 piglets (7.9 ± 1.0 kg) were used in a feeding experiment with dried oregano. The diets differed in their oregano content: 0 g, 2 g, 4 g and 8 g oregano/kg feed, corresponding to 0, 23.5, 46.9 and 93.9 mg carvacrol/kg DM. After the experimental period of 5 weeks, 20 piglets of both extreme feeding groups were slaughtered: 10 animals of the control group and 10 animals of the group that received 8 g oregano/kg. Ingesta samples of jejunum, caecum and colon were collected and analyzed by FISH and PCR RFLP to compare the diversity of microbiota. The results showed no significant changes in microbiota in response to oregano. The patterns of the PCR-RFLP showed a similarity of 61.8% - 91.8% in both feeding groups. In conclusion, an effect of oregano on the in- testinal microbiota could not be shown under the methods used.
基金supported by grants from China National Programs for Science and Technology Development (Grant No. 2003BA712A11-24)Scientific Research Fund of North China Coal Medical College (Grant No. 2005-14)
文摘Objectives Tumor necrosis factor-α (TNF-α) may play an important role in host's immune response to mycobacterium tuberculosis (M. tuberculosis) infection. This study was to investigate the association of TNF-α gene polymorphism with pulmonary tuberculosis (TB) among patients with coal worker's pneumoconiosis (CWP). Methods A case-control study was conducted in 113 patients with confirmed CWP complicated with pulmonary TB and 113 non-TB controls with CWP. They were matched in gender, age, job, and stage of pneumoconiosis. All participants were interviewed with questionnaires and their blood specimens were collected for genetic determination with informed consent. The TNF-α gene polymorphism was determined with polymerase chain reaction of restriction fragment length polymorphism (PCR-RFLP). Frequency of genotypes was assessed for Hardy-Weinberg equilibrium by chi-square test or Fisher's exact probability. Factors influencing the association of individual susceptibility with pulmonary TB were evaluated with logistic regression analysis. Gene-environment interaction was evaluated by a multiplieative model with combined OR. All data were analyzed using SAS version 8.2 software. Results No significant difference in frequency of the TNF-α-308 genotype was found between CWP complicated with pulmonary TB and non-TB controls (2,2=5.44, P=-0.07). But difference in frequency of the TNF-α-308 A allele was identified between them (2,2-5.14, P=0.02). No significant difference in frequencies of the TNF-α-238 genotype and allele (P=0.23 and P=0.09, respectively) was found between cases and controls either, with combined (GG and AA) OR of 3.96 (95% confidence interval of 1.30-12.09) at the -308 locus of the TNF-α gene, as compared to combination of the TNF-α-238 GG and TNF-α-308 GG genotypes. Multivariate-adjusted odds ratio of the TNF-α-238 GG and TNF-α-308 GA genotypes was 1.98 (95% CI of 1.06-3.71) for risk for pulmonary TB in patients with CWP. There was a synergic interaction between the TNF-a-308 GG genotype and body mass index (OR=4.92), as well as an interaction between the TNF-α-308 GG genotype and history of BCG immunization or history of TB exposure. And, the interaction of the TNF-α-238 GG genotype and history of BCG immunization or TB exposure with risk for pulmonary TB in them was also indicated. Conclusions TNF-α-308 A allele is associated with an elevated risk for pulmonary TB, whereas TNF-α-238 A allele was otherwise.
文摘A rice population consisting of 90 TN1/Guiyigu F3 lines was employed to analyze the linkage between DNA markers and a new gene Wbph6(t) conferring resistance to whitebacked planthopper, Sogatella furcifera By using the mapping approach of bulked extremes and recessive class, Wbph6(t) was mapped onto the short arm of chromosome 11 with a genetic distance of 21.2 cM to SSLP marker RM167.
基金the Science Foundation ofJiangsu Province, China (No. BK2005402)the Nation-al Natural Science Foundation of China (No. 30640018)
文摘To characterize the effects of pentachlorophenol (PCP) on the performance and microbial community of aerobic granular sludge in sequencing batch reactor (SBR), the web-based terminal restriction fragment length polymorphism (T-RFLP) and real-time PCR (RT- PCR) techniques were used to explore the bacterial community structure. When PCP increased from 0 to 50 mg/L, the COD removal rate changed little, while the ammonia removal rate dropped from 100% to 64.9%. The results of molecular characterization showed t...
基金the National NaturalScience Foundation of China (No. 39925007)the HiTech Research and Development Program (863) of China(No. 2002AA60l021)the Pilot Project of KnowledgeInnovation Program of Chinese Academy of Sciences (No.KSCX2-SW-102)
文摘The bacterial diversity of activated sludge from submerged membrane bioreactor (SMBR) was investigated. A 16S rDNA clone library was generated, and 150 clones were screened using restriction fragment length polymorphism (RFLP). Of the screened clones, almost full-length 16S rDNA sequences of 64 clones were sequenced. Phylogenetic tree was constructed with a database containing clone sequences from this study and bacterial rDNA sequences from NCBI for identification purposes. The 90.6% of the clones were a?l...
基金supported by the Research Fund from China Priority Scientific Research Project for Water Pollution Control and Treatment (No. 2008ZX07526‐001‐004)
文摘Objective To compare the bacterioplankton communities in streams exposed to pollution of different types. Methods The bacterioplankton communities in three selected heavily polluted streams were investigated by using terminal‐restriction fragment length polymorphism (T‐RFLP) analysis in combination with 16S rRNA gene clone library analysis. Results Both T‐RFLP and 16S rRNA gene clone library revealed a great difference in bacterioplankton community composition in the different streams. Conclusion This work might provide some new insights into bioremediation of heavily polluted streams.
基金the National Natural Science Foundation of China (No. 30670736 and 30500168)the Department of Science and Technology of Jiangsu Province (No. BS2006533).
文摘γ -actin (ACTG1) gene is a cytoplasmic nonmuscle actin gene, which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea. Mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families, respectively. In this study, a novel missense mutation (c.364A〉G; p.I122V) co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls. The alteration of residue Ile122 was predicted to damage its interaction with actin-binding proteins, which may cause disruption of hair cell organization and function. These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum of ACTG1 mutations causing hearing loss.
基金supported by grantsfrom the National Natural Science Fundation of China(40776085 and 40576052)State Oceanic Administration of China(908-ST-01-08-Coral Reefs Survey)Bureau of Science and Technology for Resources and Environment(YTZJJ0502)
文摘Symbiotic algae (Symbiodinium sp.) in scleractinian corals are important in understanding how coral reefs will respond to global climate change. The present paper reports on the diversity of Symbiodinium sp. in 48 scleractinian coral species from 25 genera and 10 families sampled from the Xisha Islands in the South China Sea, which were identified with the use of restriction fragment length polymorphism (RFLP) of the nuclear ribosomal DNA large subunit gene (rDNA). The results showed that: (i) Symbiodinium Clade C was the dominant zooxanthellae in scleractinian corals in the Xisha Islands; (ii) Symbiodinium Clade D was found in the corals Montipora aequituberculata, Galaxea fascicularis, and Plerogyra sinuosa; and (iii) both Symbiodinium Clades C and D were found simultaneously in Montipora digitata, Psammocora contigua, and Galaxeafascicularis. A poor capacity for symbiosis polymorphism, as uncovered by RFLP, in the Xisha Islands indicates that the scleractinian corals have low adaptability to environmental changes. Further studies are needed to investigate zooxanthellae diversity using other molecular markers.
基金supported by the 111 Project from the Ministry of Education of China (B07049)the PhD Program Foundation from the Ministry of Education of China (20100204110004)the National Natural Science Foundation of China (31371913)
文摘A novel phytoplasma was detected in a cherry plum(Prunus cerasifera Ehrh) tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies(PLBs) were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 μm, and distributed in the phloem cells of the infected tissues. A 1 246-bp 16 S ribosomal RNA(rRNA) gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16 S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows(AY) group(16SrI group). Virtual and actual restriction fragment length polymorphism(RFLP) analysis of the 16 S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group(16SrI) by four restriction sites, Hinf I, Mse I, Sau3 A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16 S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf(CPYL) symptoms is probably a distinct and novel subgroup lineage in the AY group(16SrI). In addition, the novel phytoplasma was experimentally transmitted to periwinkle(Catharanthus roseus) plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder(Cuscuta odorata) connections.
文摘Objective: To understand the role of mitochondrial DNA (mtDNA) in carcinogenesis. Methods: single-step method was used to isolate the mtDNA from human lung adenocarcinoma cell line SPC-A-1. The mtDNA was analyzed by restriction fragment length polymorphism (RFLP) with 11 kinds of restriction endonuclease, which were Pvu II, Xho I, Pst I, EcoR I, BstE II, Hind III, Hpa I, Bc1 I, EcoR V, Sca I and Xba I. Restriction map of mtDNA from SPC-A-1 cell was obtained by the single and double-digestion method. Results: It was found that no variation at 32 restriction-sites could be detected in the coding region of mtDNA from SPC-A-1 cell line. But a new site was found at nucleotide 16276 (EcoR V) within the noncoding region. Conclusion: These results indicate that the primary structure of gene coding region of mtDNA isolated from SPC-A-1 cell is highly stable. While the major variation of nucleotide is probably located in the noncoding region.
文摘After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the effect of excessive CdCl 2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl 2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl 2 did not inhibit the replication and amp + genes expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl 2 treatment for 24 hours might cause the sequences change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl 2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl 2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.
文摘DNA from 36 patients with chronic myelogenous leukemia (CML) at various clinical stages and 6 cases of acute leukemia was investigated for alterations of the p53 gene by Southern blot analysis.Rearrangements of the p53 gene were seen in 3 of 12 (25.00%) cases of blast crisis and accelerated phase (AP) of CML and in only one of 18 chronic phrase (CP),just as has been reported previously. Meanwhile,by restriction fragment length polymorphism (RFLP) analysis the Bgl II site polymorphism in the p53 gene was also found. The frequency in Chinese people detected here was 0.392,which was strikingly higher than that in some other countries(P<0. 001).These results suggested that the alterations of the p53 gene, for example,p53 rearrangements,were probably responsible for the progression of BC in some CML patients, and that the frequency of Bgl II polymorphism in the p53 gene might be related to the population distribution.
文摘Using human nov (nephroblastoma overexpressed,nov) DNA as probe,hybridization to total cellular DNAs of tumor and normal cells digested by restriction enzymes BamHI or EcoRI respectively was carried out through Southern blot. It was observed that nov gene in these cells is not only highly conserved, but also certains RFLP characteristic . The correlation between RFLP characteristic of nov gene and its function was analyzed .
文摘The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g<sup>-1</sup>, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g<sup>-1</sup>. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g<sup>-1</sup>. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g<sup>-1</sup>) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g<sup>-1</sup>), and the other Firmicutes (3.7 × 106 MPN g<sup>-1</sup>). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g<sup>-1</sup>), Burkhokderia sp. (1.5 × 106 MPN g<sup>-1</sup>), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g<sup>-1</sup>). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.
文摘Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 10<sup>6</sup> MPN g<sup>-1</sup> dry soil), followed by γ-Proteobacteria (1.32 × 10<sup>6</sup> MPN), δ-Proteobacteria (0.006 × 10<sup>6</sup> MPN), and the other gram negative bacteria (0.006 × 10<sup>6</sup> MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 10<sup>6</sup> MPN), followed by α-Proteobacteria (26.0 × 10<sup>6</sup> MPN), β-Proteobacteria (17.1 × 10<sup>6</sup> MPN), δ-Proteobacteria (11.2 × 10<sup>6</sup> MPN), the other Firmicutes (1.71 × 10<sup>6</sup> MPN), γ-Proteobacteria (0.5 × 10<sup>6</sup> MPN), and the other gram negative bacteria (0.05 × 10<sup>6</sup> MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 10<sup>6</sup> MPN), followed by Actinobacteria (2.14 × 10<sup>6</sup> MPN), Bacillus sp. (2.14 × 10<sup>6</sup> MPN), and γ-Proteobacteria (0.35 × 10<sup>6</sup> MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candidate of a new evaluation system of soil and manure.
文摘In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L<sup>-1</sup><sup></sup>) were included in row cattle feces (1.78 × 10<sup>4</sup> MPN g<sup>-1</sup>) and cattle feces manure (>3.84 × 10<sup>4</sup> MPN g<sup>-1</sup>). Compost originated from leftover food (>44.8 × 10<sup>4</sup> MPN g<sup>-1</sup>) and shochu lee (>320 × 10<sup>4</sup> MPN g<sup>-1</sup>) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L<sup>-1</sup><sup></sup>), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 10<sup>4</sup> MPN g<sup>-1</sup>), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L<sup>-1 </sup>of<sup> </sup>ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 10<sup>4</sup> MPN g<sup>-1</sup>), followed by cattle feces manure (4.19 × 10<sup>4</sup> MPN g<sup>-1</sup>), and shochu lee (0.36 × 10<sup>4</sup> MPN g<sup>-1</sup>), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.
基金ThisprojectwassupportedbytheZhejiangProvincalNaturalScienceFoundation (No 39842 6 )
文摘Objective To establish the specific 16S-23S rRNA gene spacer regions in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis. Methods A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA from sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 26 species was amplified by PCR, and further analyzed by RFLP, DNA cloning and sequences analysis. Furthermore, all specimens were examined by bacterial culturing and PCR-RFLP analysis. The evaluation of these assays in practical clinic practice was also discussed.Results Restriction enzyme analysis revealed one, two or three bands or more observed among the 26 different standard strains. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction with human genomic DNA, fungus or virus. Fourteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf Ⅰ or Alu Ⅰ digestion. The only difference between K.pneumoniae and E.durans was located at the site of the 779th nucleotide according to the sequence analysis and only XmaⅢ digestion could distinguish one from another. Of 42 specimens from septicemic neonates, 15 were identified as positive by blood culture at a rate of 35.7%. However, 27 specimens identified as positive by PCR, with a rate of 64.2%, a method significantly more effective than blood culture (P<0.01). Of 6 cerebrospinal fluid (CSF) specimens, one tested positive for S.epidermidis was also positive by PCR, two culture negative were positive by PCR and diagnosed as S.epidermidis according to the DNA pattern. One positive for C.neoformans was negative by PCR. The other two specimens were negative by both PCR and culture.Conclusions The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in providing a new technique for detecting pathogens in clinical bacterial infections.