Dynamic changes of telomeric restriction fragment (TRF) lengths in cells during the developmental process from embryos to seedlings and a comparison with the embryonal calli in Ginkgo biloba L.

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.

Comparing the frequencies of restriction fragment length polymorphisms for dystrophin gene in Chinese with those from Japanese and Caucasian populations

The restriction fragment length polymorphisms distribution and frequency of dystrophin gene in Chinese were studied by using 14 subclones of the entire 14kb cDNA for the dystrophin as hybridization probes. Allelic fragments were detected in hybridization patterns of PvuII/1a, Taq I/2b-3, Taq I/5b-7, and Xba I/10. Among them, the allelic fragments (26kb and 3.8kb) in PvuII/2b-3 pattern and the allelic fragments (10.0kb and 8.4kb) in Taq I/5b-7 patterns had never been reported previously. Compared with the data from Caucasians and Japanese, it indicated that there was a significant difference (P<0.01) of the allelic fragment frequency in Taq I/2b-3 and Xba I/10 patterns between Chinese and Caucasians. The frequencies of allelic fragments A2 (5.6kb) in Taq I/8 and A2 (10.7kb) in EcoR V/9 were high in Caucasians, yet had not been detected in Chinese, the differences were also highly significant. But in Chinese and Caucasians, the B1B2 allelic frequencies in Taq I/5b-7 are the same. As to the frequency of the allelic fragments A1A2 and B1B2 in Pvu II/1a, there was no significant difference between Chinese and Japanese.

Evaluation of the Method Based on Restriction Fragment Length Polymorphism Analysis as Simple Analysis Method of Lactic Acid Bacteria in Foods

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-1) 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.

The Use of Restriction Fragment Length Polymorphism and Fluorescence in Situ Hybridization to Investigate Microbiota of Piglets after Feeding Oregano

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.

Analysis of polymorphisms in the promoter region of interleukin-1 β by restriction fragment length polymorphism-PCR

Objective: To investigate the frequencies of -1470, (-511) and -31 single nucleotide polymorphisms (SNPs) in the promoter of IL-1β and its haplotype constitution in Chongqing population. Methods: One hundred and twelve healthy Chongqing people were enrolled in this study. Polymorphisms at -1470 (G to C), (-511) (T to C) and -31 (C to T) of IL-1β were genotyped with the method of restriction fragment length polymorphism (RFLP). Haplotype frequencies were analyzed by Arlequine software.Results: Frequencies of IL-1β -1470, -511 and -31 SNPs were (41.67)%, 50% and (45.33)%, respectively. Genotype frequencies of -1470 locus were (39.81)%, (37.04)% and (23.15)% for G/G, G/C and C/C respectively. As for T-511C SNP, genotype frequencies of (T/T), T/C and C/C were (29.91)%, (40.18)% and (29.91)%, respectively. Genotyping results of C/C, C/T, and T/T of -31 locus were (35.51)%, (38.32)% and (26.71)% respectively. Haplotype analysis found that there were mainly three haplotypes constituted by three SNPs, ie., G-T-C, C-T-C and G-C-T. Conclusions: Polymorphisms exist in the promoter of IL-1β in Chongqing population. Three SNPs locate in the same haplotype block.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM（RFLP） ANALYSIS OF GENOMIC DNA OF 5 STRAINS OF TRICHINELLA SPIRALIS IN CHINA

Five restriction endonucleases were used to digest genomic DNA from 5 isolates of Trichinella spiralis obtained from Changchun, Tianjin, Xian, Henan and Yunnan. All the isolates were secured from pigs except the Changchun strain which came from dog. The DNA fragments digested by endonuclease were separated by agarose gel electrophoresis. The Changchun isolate had a EcoRI band at 1. 12kb and a DraI band at 1. 97kb which were unique to this isolate. A cloned specific repetitive DNA sequence (1. 12kb) from the Changchun strain was selected to prepare a probe for the Southern blotting of EcoRI restriction DNA fragments for the 5 isolates. The 1.12kb hybridizing band did not appear except in the Changchun isolate.These results seem to indicate that there are differences between the isolates obtained from hosts in different geographical regions.

RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIALDNA FROM HUMAN LUNG ADENOCARCINOMA CELLLINE SPC-A-1

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.

RFLP Detection of Genetic Variation of Maize Inbred Lines

Genetic similarities of 13 inbred lines of maize (Zea mays L.) were analyzed by restriction fragment length polymorphisms (RFLPs). The objectives of the study were to detect genetic similarities among 13 inbreds and to assign them to heterotic groups. By means of 24 probe_enzyme combinations (PECs) selected for locus specificity, clear patterns and reproducibility, 85 alleles were found with an average of 3.3 alleles per locus. The allelic frequency data were used to estimate genetic similarities among lines, and as a result the diversity index of 0.499 was obtained. Genetic similarities between the pairs of 13 lines ranged from 0.523 up to 0.802 with an average of 0.649. The UPGMA clustering algorithm analysis classified the 13 lines into five groups, which generally corresponded to known maize heterotic groups based on pedigree information. The authors concluded that RFLP_based markers could be used for investigating genetic relationships between maize inbred lines and assigning them to heterotic groups, but it seemed that a large number of PECs were needed to obtain reliable estimates of genetic similarity.

Inheritance of Chloroplast and Mitochondrial DNA in Chinese Fir (Cunninghamia lanceolata)

The inheritance of mitochondrial (mt) DNA and chloroplast (cp) DNA was investigated in intergeneric hybrids from crossing between Cunninghamia lanceolata (Lamb.) Hook. and Cryptomeria fortunei Hooibrenk. The chloroplast trnL trnF region and one intra genic segment of the mitochondrial gene, Cox Ⅲ, were amplified from those of the parents and hybrids by PCR using gene specific primers. Cp and mtDNA polymorphisms of the amplified regions were detected between the parents after restriction digestions. Restriction fragment length polymorphism (RFLP) analysis revealed that all the F 1 individuals possessed Cox Ⅲ restriction fragment patterns (characteristic of the paternal parent Cryptomeria fortunei ) and the trnL trnF region (identical to the maternal parent Cunninghamia lanceolata ) showing that a different mode of inheritance for organelle DNA has occurred in the hybrids. Furthermore, the maternal inheritance of chloroplast DNA is reported here for the first time in coniferophyta.

Studies on the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer

AIM To study the relationship between the point mutation of ras oncogenes and the prognosis of patients with gastric cancer.

The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province,a non HCC prevalent area in China

AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.

Association of TNF-α-238G/A and 308 G/A Gene Polymorphisms with Pulmonary Tuberculosis among Patients with Coal Worker’s Pneumoconiosis

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.

Mapping of a New Gene Wbph6(t) Resistant to the Whitebacked Planthopper, Sogatella furcifera, in Rice

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.

Clinical significance of telomerase and its associate genes expression in the maintenance of telomere length in squamous cell carcinoma of the esophagus

AIM: To observe the interaction between the expression of telomerase activity (TA) and its associate genes in regulation of the terminal restriction fragment length(TRFL) in esophageal squamous cell carcinoma (SCC).METHODS: Seventy-four specimens of esophageal SCC were examined. The TA was measured by telomeric repeat amplification protocol (TRAP) assay, and the associated genes [human telomerase-specific reverse transcriptase (hTERT), hTERC, TP1, c-Myc, TRF1,and TRF2] were detected using RT-PCR method. The TRFL was measured by Telomere Length Assay Kit and Southern blotting. The correlations between the expression of telomerase and its associated genes with the TRFL and survivals were examined.RESULTS: Expressions of the TA, hTERT, hTERC, TP1,c-Myc, TRF1, and TRF2 genes were observed in 85.1%,64.9%, 79.7%, 100.0%, 94.6%, 82.4%, and 91.9% of the tumor tissues, respectively. The TRFL of the tumor and normal esophageal tissues were 2.70±1.42 and 4.93±1.74 kb, respectively (P＜0.0001). The TRFL of the telomerase positive and telomerase negative tumor tissues were 2.72±1.44 and 2.58±1.32 kb, respectively (P = 0.767).The TRFL ratios (TRFLR) of the telomerase positive and telomerase negative tumor tissues were 0.55±0.22 and 0.59±0.41, respectively (P = 0.742). The expression rates of h-TERT (P = 0.0002), hTERC (P＜0.0001), and TRF1(P = 0.002) in the tumor tissues are higher than those of the normal paired tissues. Though TA is markedly activated in tumor tissues (P＜0.0001), its expression is not related to clinicopathological parameters including gender, tumor differentiation, and TNM stages. The cumulative 4-year survival rates of telomerase positive and telomerase negative cases were 35.86% and 31.2%,respectively (P = 0.8442). The cumulative 4-year survival rates of patients with their TRFLR ≤85% and ＞85%were 38.7% and 15.7%, respectively (P = 0.1307).CONCLUSION: Though telomerase expression is not related to tumor stages and prognosis, our data support that the TA increased as the TRFL decreased,probably under the control of hTERT, hTERC, and TRF1.When telomerase expression was activated, only TRF2overexpression persisted to stabilize T-loop formation.Furthermore, as the TRFLR decreased to 85%, a trend of better prognosis was observed. Cox model analysis indicates a higher t/n TRFLR and distant metastasis are independent poorer prognostic factors (P = 0.035 and P = 0.042, respectively).

Torque teno virus:Its prevalence and isotypes in North India

AIM： To investigate the prevalence and genotype distribution of Torque teno virus （TTV） in patients with different liver diseases and chronic renal failure treated at a referral hospital in North India. METHODS： Whereas prevalence of TFV was based on amplification of conserved region of ORF2 of TTV genome, the genotyping of TFV was carried out using restriction fragment length polymorphism （RFLP） procedure on the N22 region of ORFI. RESULTS： TTV-DNA was detected in 137 of 513 （26.7%） patients with liver diseases and 38 of 65 （58.5%） patients with chronic renal failure. Trv was also detected in 2/7% of healthy controls. The sequence analysis of the PCR product from 10 randomly selected cases failed to show a significant sequence divergence when compared with that of the TRM1 isolate of TTV genotype 1. The results of genotyping in 55 randomly selected patients showed the presence of genotype 1 （G1） in 53 （96.4%） and genotype 2 （G2） in 2 cases （3.6%）, respectively. Other genotypes were not identified in this patient subgroup, suggesting that G1 is predominant in this area. The results of genotyping by RFLP were also supported by phylogenetic tree analysis, where G1 was found to be the major genotype. CONCLUSION： These results indicate that TTV is moderately present in Indian patients, with G1 to be the major genotype in North India. The pathogenicity and etiological role of TTV in different diseases is still a question mark and warrant further studies.

Effect of herbal medicine Juzentaihoto on hepatic and intestinal heat shock gene expression requires intestinal microflora in mouse

AIM： To evaluate the role of intestinal microflora in the effects of multi-herbal medicine on gene expression in the gut and liver. METHODS： The multi-herbal medicine Juzentaihoto （JTX） was administered to five germ-free mice and regular mice for 2 wk. Among the results of the comprehensive gene chip analysis of the intestine and liver, we featured heat shock proteins （HSPs） 70 and 105 because their gene expression changed only in the presence of microflora. Real-time RT-PCR was performed to confirm the expression levels of these HSP genes. To determine whether JTX acts directly on the HSP genes, sodium arsenite （SA） was used to induce the heat shock proteins directly. To examine the change of the intestinal microflora with administration of JTX, the terminal restriction fragment polymorphism （T-RFLP） method was used. To identify the changed bacteria, DNA sequencing was performed.documented by gene chip and real-time RT-PCR, changed with the administration of JTX in the regular mice but not in the germ-free mice. JTX did not suppress the direct induction of the HSPs by SA. T-RFLP suggested that JTX decreased unculturable bacteria and increased Lactobacillus johnsoni. These data suggested that JTX changed the intestinal microflora which, in turn, changed HSP gene expression.CONCLUSION： Intestinal microflora affects multi-herbal product JTX on the gene expression in the gut and liver.

Molecular characterization of bacterial community in aerobic granular sludge stressed by pentachlorophenol

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...

Bacterial diversity in activated sludge from a consecutively aerated submerged membrane bioreactor treating domestic wastewater

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...

POLYMORPHISMS OF THE HUMAN LIPOPROTEIN LIPASE GENE:POSSIBLE ASSOCIATION WITH LIPID LEVELS IN PATIENTS WITH CORONARY HEART DISEASE IN BEIJING AREA

The polymorphisms(Pvu Ⅱand Hind Ⅲ) on the lipoprotein lipase(LPL) gene locus was investigated in a sample of 100 patients surviving previous myocardial infarction and 100 age matched healthy individuals selected from Han Chinese of Beijing area.In patient group a strong association was found between H+allele of Hind Ⅲ polymorphism and raised TG levels(P<0.01).In control group P-P-genotype was observed to be associated with higher TG levels compared with P+P genotype of Pvu Ⅱ polymorphism(P<0.05).Combination of H+H+ genotype with P-P-genotype showed the highest TG levels among all nine kinds of genotype combinations in patient group(P<0.01).However,comparison of distribution of alleles and genotypes of these polymorphisms between patient group and control group demonstrated no significant difference. Our data suggest that the polymorphisms at the LPL gene,as the linkage markers with an aetiologic mutation at or around LPL gene,may constitute one of the genetic determinants for the population variation in plasma TG levels,as well as for the common dyslipidemia in Chinese population.