DNA sequence representation by trianders and determinative degree of nucleotides

A new version of DNA walks, where nucleotides are regarded unequal in their contribution to a walk is introduced, which allows us to study thoroughly the “fine structure” of nucleotide sequences. The approach is based on the assumption that nucleotides have an inner abstract characteristic, the determinative degree, which reflects genetic code phenomenological prop-erties and is adjusted to nucleotides physical properties. We consider each codon position independently, which gives three separate walks characterized by different angles and lengths, and that such an object is called triander which reflects the “strength” of branch. A general method for identifying DNA sequence “by triander” which can be treated as a unique “genogram” (or “gene passport”) is proposed. The two- and three-dimensional trianders are considered. The difference of sequences fine structure in genes and the intergenic space is shown. A clear triplet signal in coding sequences was found which is absent in the intergenic space and is independent from the sequence length. This paper presents the topological classification of trianders which can allow us to provide a detailed working out signatures of functionally different genomic regions.

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

Diversity Analysis in Selected Non-basmati Scented Rice Collection

Diversity analysis among 23 rice varieties including 16 non-basmati scented accessions, 5 basmati accessions and 2 non-scented accessions was performed by random amplified polymorphic DNA （RAPD） and inter-simple sequence repeat （ISSR） marker systems. The varieties analyzed by 11 RAPD and 8 ISSR primers yielded an average of 65% and 80% polymorphism, respectively. The average number of polymorphic bands generated per RAPD primer was 6 and per ISSR primer was 5.87. RAPD and ISSR data analysis individually could not segregate basmati and non-basmati scented rice accessions. However, the analysis using a combined data could group basmati and non-basmati scented rice accessions separately. The bands present specifically among three accessions of non-basmati scented rice were also identified. The study revealed a high genetic diversity among non-basmati scented rice accessions.

Isolation and Characterization of Microsatellites in Snap Bean

The objectives of this study were to isolate and characterize microsatellites from a heat tolerant variety of snap bean (Phaseolus vulgaris L.) in order to generate polymorphic genetic markers linked to quantitative trait loci for heat tolerance. A genomic library contained 400-800 bp inserts was constructed and screened for the presence of (GA/CT) n and (CA/GT) n repeats. The proportion of positive clones yielded estimated of 3.72×10 4 such dinucleotide repeats per genome, roughly comparable to the abundance reported in other eukaryotic genomes. Twenty_six positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) n motif was much more abundant than the (CA/GT) n motif in these clones. The (GA/CT) n repeats also showed longer average repeat length (mean n =10.4 versus 6.5), suggesting that they are better candidates for yielding polymorphic genetic markers in the snap bean genome.

Molecular characterization and subtyping of Blastocystis in urticarial patients in Turkey

Objective:To investigate Blastocystis’etiologic role and association with gastrointestinal symptomatology in acute and chronic urticaria patients and to identify Blastocystis subtypes responsible for urticaria.Methods:The study included urticaria patients and healthy individuals that presented to our polyclinic between June 2015 and May 2017.The participants were assigned into Group栺(137 patients),subdivided into acute(72)and chronic urticaria patients(65),and Group栻(129 control individuals).Blastocystis presence was investigated by native-Lugol examination,trichrome staining,PCR using sequence tagged site primers,and DNA sequencing analysis.The phylogenetic tree was constructed.Results:The native-Lugol and trichrome staining methods revealed that 16 patients(16/133,12.0%)had Blastocystis-positive stool samples,of which seven samples(7/133,5.3%)belonged acute and nine(9/133,6.8%)to chronic urticaria patients.Concerning Blastocystis subtypes,of the acute urticaria patients,three had subtype 1(ST1),one had ST2,and three had ST3.Of the chronic urticaria patients,one had ST1 and eight had ST3.Blastocystis positivity was detected in two control individuals(2/123,1.6%),both being ST3.All subtypes identified by PCR were confirmed by the sequencing analysis.The acute and chronic urticaria groups showed no statistically significant differences for Blastocystis positivity(P=0.60)and subtype distribution(P=0.15).A statistically significant difference was found between the urticaria patients and the controls for Blastocystis positivity(P<0.01),but not for subtype distribution(P=0.67)or for Blastocystis presence and gastrointestinal complaints.Conclusions:This study on Blastocystis subtype distribution among Turkish urticaria patients showed results consistent with the literature.It was concluded that Blastocystis should be kept in mind in patients with urticaria.

Molecular characterization of Acidithiobacillus ferrooxidans strains isolated from different environments by three PCR-based methods

PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from different environments.(GTG)5 and BOXA1 R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles from A. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains of A. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using(GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16 S r DNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a new Acidithiobacillus named Acidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.

Difference in DNA sequences in SSU rDNA variable regions among pathogens isolated from different epidemic foci of visceral leishmaniasis in China

To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L d ) isolates from different epidemic foci in China Methods Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM R T Easy Vectors After that, the specific fragments were sequenced by an automated DNA sequencer Results Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length All 5 point mutations were located in two unique sequence blocks (UQ Ⅰ and UQ Ⅱ), and no insertions or deletions were found The identities of comparison of Leishmania in GeneBank were more than 98% Conclusion Five point mutations exist in the SSU rDNA variable region of 5 L d isolates from different epidemic foci of visceral leishmaniasis (VL) in China Sequence differences of the SSU rDNA variable region exist among L d isolates from different foci

Cloning of differentially expressed genes in human hepatocellular carcinoma and nontumor liver

INTRODUCTIONThe mechanism of hepatocellular carcinoma(HCC)is still unclear,although some genes have been found to play a role in the transformation of liver cells,and a variety of studies have described differences in gene expression which distinguished tumor from nontumor[1-6].The new genes,especially the functional genes directly related with tumor are still worth being found.The purpose of our study is to find the different genes between human liver tumor and normal tissues using suppression subtractive hybridization.

High frequencies of HGV and TTV infections in blood donors in Hangzhou

AIM: To determine the frequencies of HGV and TTV infections in blood donors in Hangzhou. METHODS: RT-nested PCR for HGV RNA detection and semi-nested PCR for TTV DNA detection in the sera from 203 blood donors, and nucleotide sequence analysis were performed. RESULTS: Thirty-two (15.8%) and 30 (14.8%) of the 203 serum samples were positive for HGV RNA and TTV DNA, respectively. And 5 (2.5%) of the 203 serum samples were detectable for both HGV RNA and TTV DNA. Homology of the nucleotide sequences of HGV RT-nested PCR products and TTV semi-nested PCR products from 3 serum samples compared with the reported HGV and TTV sequences was 89.36%, 87.94%, 88.65% and 63.51%, 65.77% and 67.12%, respectively. CONCLUSION: The infection rates of HGV and/or TTV in blood donors are relatively high, and to establish HGV and TTV examinations to screen blood donors is needed for transfusion security. The genomic heterogeneity of TTV or HGV is present in the isolates from different areas.

Genetic instability in cancer tissues analyzed by random amplified polymorphic DNA PCR

OBJECTIVE: To detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. METHODS: Five kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned, labeled as probes for Southern and Northern blot analysis and DNA sequencing. RESULTS: Sample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40% (42.2% - 49.4%). There were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% (primer 8) to 68% (primer 2). Band B is a single copy fragment, and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005. Further analysis revealed that it might be part of a cis-regulatory element of a new tumor suppressor gene, containing a promoter of cis-action 'CACA' box, an enhancer of 'GATA' family and a start codon. CONCLUSIONS: It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.

Two novel mutations of the retinitis pigmentosa GTPase regulator gene in two Chinese families with X-linked retinitis pigmentosa

Objective To detect mutations of the retinitis pigmentosa GTPase regulator (RPGR) gene in two Chinese X-linked retinitis pigmentosa families. Methods Fragments of exons 1-19 of the RPGR gene were amplified with intronic primers, using genomic DNA as template. The polymerase chain reaction (PCR) products were analysed by single-strand conformation polymorphism (SSCP) and direct sequencing. Mutations were identified by comparing DNA sequences of the patients with those of the normal controls.Results Two novel mutations, c1536delC and E332X, were identified in exons 12 and 9 of the RPGR gene in both families. Each mutation was the first mutation found in their respective exons. Both mutations were predicted to cause premature termination, which resulted in truncated proteins without normal functions of the RPGR products.Conclusions Both mutations are the genetic basis of the pathogenesis in the respective families. Our data might be helpful in analysing the function of the RPGR protein.

The intraspecific difference of the triose phosphate isomerase (tim) gene from Giardia lamblia

OBJECTIVE: To investigate the intraspecific difference of the triose phosphate isomerase (tim) gene from Giardia lamblia (G. lamblia). METHODS: Total genomic DNA of G. lamblia was extracted and partial fragments of the triose phosphate isomerase (tim) gene were amplified by polymerase chain reaction (PCR). All nucleotide sequences were analyzed by using a phylogenetic analysis, which was constructed with parsimony and Neighbor-joining (N-J) methods. RESULTS: A total of 124 variable sites (23% of all sequences detected) was defined, most of which were found at the silent sites of codons. Two similar phylogenetic trees were constructed, subdividing 16 Giardia isolates into two groups. CONCLUSION: The genetic diversity of G. lamblia appeared to be little affected by factors of both host and geography, while natural-selection played an important role in DNA molecular evolution level of the tim gene. The tim gene may be considered a very useful genetic marker of the population genetic structure of G. lamblia.

Continuous desulfurization and bacterial community structure of an integrated bioreactor developed to treat SO_2 from a gas stream

Sulfide dioxide（SO2） is often released during the combustion processes of fossil fuels. An integrated bioreactor with two sections, namely, a suspended zone（SZ） and immobilized zone（IZ）, was applied to treat SO2 for 6 months. Sampling ports were set in both sections to investigate the performance and microbial characteristics of the integrated bioreactor. SO2 was effectively removed by the synergistic effect of the SZ and IZ, and more than 85%removal efficiency was achieved at steady state. The average elimination capacity of SO2 in the bioreactor was 2.80 g/（m3·hr） for the SZ and 1.50 g/（m3· hr） for the IZ. Most SO2 was eliminated in the SZ. The liquid level of the SZ and the water content ratio of the packing material in the IZ affected SO2 removal efficiency. The SZ served a key function not only in SO2 elimination, but also in moisture maintenance for the IZ. The desired water content in IZ could be feasibly maintained without any additional pre-humidification facilities. Clone libraries of 16 S r DNA directly amplified from the DNA of each sample were constructed and sequenced to analyze the community composition and diversity in the individual zones.The desulfurization bacteria dominated both zones. Paenibacillus sp. was present in both zones, whereas Ralstonia sp. existed only in the SZ. The transfer of SO2 to the SZ involved dissolution in the nutrient solution and biodegradation by the sulfur-oxidizing bacteria.This work presents a potential biological treatment method for waste gases containing hydrophilic compounds.

Renal cell carcinoma related novel gene, GYLZ-RCC18: cloning and functional studies

OBJECTIVE: To clone the full length of renal cell carcinoma (RCC) related novel gene GYLZ-RCC18 and study its function. METHODS: SMART RACE technology was used to clone the full length of GYLZ-RCC18. RT-PCR was used to detect its expression in renal cell carcinoma tissue at different stages and grades. We transfected the antisense oligonucleotide of GYLZ-RCC18 to renal cell carcinoma cell line, GRC-1, and analyzed proliferation activity, growth rate, apoptosis, and mortality changes. RESULTS: The full length of GYLZ-RCC18 (GenBank accession number: BE825133) cDNA was about 3.5 kb. GYLZ-RCC18 had a higher expression in higher grades and stages of renal cell carcinoma than in lower ones. The expression of GYLZ-RCC18 in renal cell carcinoma was much higher than in normal kidney. After the transfection of GYLZ-RCC18 antisense oligonucleotide, the mortality of GRC-1 increased significantly, while proliferative activity and growth rate were substantially inhibited at the same time. The antisense oligonucleotide induced apoptosis of GRC-1 through the entire observation time. CONCLUSION: GYLZ-RCC18 is an important novel gene related to renal cell carcinoma. Overexpression of this gene results in higher growth and proliferative activity and has an antiapoptosis effect on renal cell carcinoma cells. Transfection of the antisense oligonucleotide may inhibit the generation and development of renal cell carcinoma.

Polymorphisms and functions of the aldose reductase gene 5' regulatory region in Chinese patients with type 2 diabetes mellitus

OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. RESULTS: Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P

The complete mitochondrial genome of Oreta fuscopurpurea(Lepidoptera: Drepanidae) and its phylogenetic implications

In this study, the first complete mitochondrial genome（mitogenome） of lepidopteran family Drepanidae（superfamily Drepanoidea） was reported with the notes about its phylogenetic implications. The Oreta fuscopurpurea mitogenome is 15,564 bp in length, including 13 protein-coding genes, two ribosomal RNAs（lrRNA and srRNA）, 22 transfer RNAs and a non-coding control region（AT-rich region）, with a 79.6% A＋T content. The gene orientation and arrangement of the mitogenome are the same as other sequenced lepidopterans. All protein-coding genes usually start with the common ATN codon except for COI gene, which used CGA as the initial codon; eight PCGs use a typical stop codon of TAA, whereas the remaining PCGs use incomplete stop codon of T or TA. All tRNAs have the typical clover-leaf structure with the exception of t RNA^（Ser）（AGN）. The lrRNA and sr RNA genes are 1,409 bp and 778 bp in size respectively, with the former harboring one（TA）_（13） microsatellite-like repeat and an 17 bp insertion. The 20 intergenic spacers totaling of 184 bp and 8 overlapping sequences totaling of 25 bp are scattered throughout the whole mitogenome. The 526 bp AT-rich region contains some structures characteristic of lepidopterans, such as the motif ATAGA preceded by an 19 bp poly-T stretch, a tRNA-like and a sterm-loop structures. Phylogenetic analysis of the Oreta fuscopurpurea with other 47 insect species covering 20 lepidopteran families were conducted based on the sequence data of the 13 mitogenomic protein coding genes with maximum likelihood（ML） and Bayesian inference（BI） methods, and the results showed distinctly that the superfamily Drepanoidea was sister to the clade of（Bombycoidea, Lasiocampoidea） ＋（Noctuoidea, Geometroidea）.