Restriction Enzymes MspI Research Articles

The diversity of bean nodule bacteria in Ukrainian agrocenoses

The aim of our work was to study the diversity of beans nodule bacteria in the natural populations of rhizobia and spend their serological and genetic typing. Beans rhizobia were isolated from bean nodules which were grown in soil samples selected in different regions of Ukraine. Morphological, cultural and physiological properties of rhizobia were studied using standard methods. The diversity of beans nodule bacteria was assessed using a serological method. The restriction analysis of the intergenic region of the ribosomal cluster (ITS) was carried out with the use of restriction endonucleases MspI, HaeIII, NdeII. Fifty strains of beans nodule bacteria were isolated from soils of different regions of Ukraine. Biological diversity of these strains was studied. All the culture of nodule bacteria enters into a symbiotic relationship with the common beans and forms active nodules on the plant roots. These strains were identified as Rhizobium phaseoli. It is established that new strains of R. phaseoli differ in antibiotics sensitivity, serological and genetic properties. On antigenic com- position investigated nodule bacteria were combined into different serological groups. The beans rhizobia, isolated from agrocenosis of Chernihіv region was the most serologically diverse. PCR-RFLP of the ITS- region showed significant genetic polymorphism of common beans microsymbionts, isolated from local populations of rhizobia. On the basis of the restriction profiles of the 16S-23S rDNA these strains were first divided into different IТS-types. It is established that division the strains on ITS-types (NdeII and HaeIII) corresponds to their division into different serological groups.

Use of cost effective and rapid molecular tools for identification of Candida species, opportunistic pathogens.

Background and Purpose :Candidiasis is a widespread fungal infection caused by different Candida species. Rapid identification of Candida species in clinical laboratory is becoming increasingly important since the identification and discrimination of ethological agents for early treatment. We aimed at molecular identification of commonly Candida species isolated from clinical samples by using both PCR-RFLP assay and amplification of hwp1 gene. Materials and Methods:Clinical samples comprising of vaginal specimens ,cutaneous, sputum, bronchoalveolar lavage(BAL,( and blood cultures were recovered from suspected patients. Candida isolates were initially identified phenotypically and confirmed by molecular approaches based on restriction fragment length polymorphism (PCR-RFLP (with MspI restriction enzyme. Amplification of hwp1 gene was performed for discrimination of C. albicans from C. dubliniensis and C. africana. Results:The most abundant species were C. albicans (n=67; 44.6 %), C. glabrata (n=10; 20 %), C. tropicalis (n=20; 13.3 %), C. krusei (n=12; 8 %), C. parapsilosis (n=11; 7.3 %). Out of 67 C. albicans species, 6 species identified as C. dubliniensis and 4 species identified as C. africana. Conclusion:High frequency of non-albicans Candida species and differences in levels of susceptibility to the antifungal agents are important issues in medicine .Therefore, to manage the Candida-related infections properly, molecular diagnostic methods would be fast, reliable and even cost-effective approaches for identification of Candida species.

Association of single nucleotide polymorphisms in genes coding insulin-like growth factor 1 system and milk production traits in Montbeliarde cows

The insulin-like growth factor 1 system plays a central role in the growth and development of the mammary gland. Insulin-like growth factor 1 (IGF1) and insulin-like growth factor 1 receptor (IGF1R) have been proposed as candidate genes for milk production traits. This study involved a population of 163 Montbeliarde cows. Five polymorphic sites were analysed using the polymerase chain reaction: restriction fragment length polymorphism (PCR-RFLP) (TaiI and MspI restriction enzymes) and amplification-created restriction site (ACRS) (SnaBI, TasI and TaqI restriction enzymes). The frequencies of the most common alleles were 0.67 for the T allele (IGF1/SnaBI), 0.85 for the A allele (IGF1/TasI), 0.95 for the C allele (IGF1R/TaiI), 0.84 for the G allele (IGF1R/MspI) and 0.69 for the G allele (IGF1R/TaqI). In the first lactation, IGF1/TasI polymorphisms and all single nucleotide polymorphisms in the IGF1R gene were associated with differences in milk, fat and protein yields, without further confirmation in the second lactation. No differences were found in milk production traits between IGF1/SnaBI genotypes and combined genotypes. To date, this is the first association study based on polymorphisms of the primary genes encoding the IGF-1 system in a small herd of Montbeliarde cows. If specific haplotypes could be determined in large-scale studies, based on Montbeliarde and other dairy breeds, it would provide a valuable genetic tool to identify causative mutations. ______________________________________________________________________________________ Keywords : IGF1, IGF1R, milk production traits, polymorphism

Construction of a high-density, high-quality genetic map of cultivated lotus (Nelumbo nucifera) using next-generation sequencing.

BackgroundThe sacred lotus (Nelumbo nucifera) is widely cultivated in China for its edible rhizomes and seeds. Traditional plant breeding methods have been used to breed cultivars with increased yields and quality of rhizomes and seeds with limited success. Currently, the available genetic maps and molecular markers in lotus are too limited to be useful for molecular genetics based breeding programs. However, the development of next-generation sequencing (NGS) technologies has enabled large-scale identification of single-nucleotide polymorphisms (SNPs) for genetic map construction. In this study, we constructed an SNP-based high-density genetic map for cultivated lotus using double digest restriction site-associated DNA sequencing (ddRADseq).ResultsAn F2 population of 96 individuals was derived from a cross between the rhizome lotus cultivar ‘Juwuba’ (male parent) and the seed lotus cultivar ‘Mantianxing’ (female parent). Genomic DNAs from this population were digested with the restriction enzymes EcoRI and MspI and then sequenced. In total, 133.65 Gb of raw data containing 1,088,935,610 pair-end reads were obtained. The coverage of reads on a reference genome was 7.2 % for the female parent, 6.56 % for the male parent, and 1.46 % for F2 individuals. From these reads, 10,753 valid SNP markers were used for genetic map construction. Finally, 791 bin markers (so-segregated adjacent SNPs treated as a bin marker), consisting of 8,971 SNP markers, were sorted into 8 linkage groups (LGs) that spanned 581.3 cM, with an average marker interval of 0.74 cM. A total of 809 genome sequence scaffolds, covering about 565.9 cM of the wild sacred lotus genome, were anchored on the genetic map, accounting for 70.6 % of the genome assembly.ConclusionsThis study reports the large-scale discovery of SNPs between cultivars of rhizome and seed lotus using a ddRADseq library combined with NGS. These SNPs have been used to construct the first high-density genetic map for cultivated lotus that can serve as a genomic reference and will facilitate genetic mapping of important traits in the parental cultivars.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2781-4) contains supplementary material, which is available to authorized users.

Identification of Candida species in the oral cavity of diabetic patients.

Background and Purpose:Diabetic patients are more susceptible to oral candidiasis infection than non-diabetics due to the factors promoting oral carriage of Candida. Several factors can increase colonization of Candida species in the oral cavity such as xerostomia, which reduces the salivary flow and is a salivary pH disorder. In the current study, we aimed to identify and compare the colonization level of Candida spp. in the oral cavity of diabetic and non-diabetic groups. Materials and Methods:Swabs were taken from the mouth of 106 participants and were cultured on Sabouraud dextrose agar (SDA) medium. Likewise, the saliva samples were collected for salivary glucose and pH measurements. The study was performed during June 2014-September 2015 on two groups of diabetic patients (n=58) and non-diabetics (n=48) as the control group. The Candida spp. were identified with PCR-restriction fragment length polymorphism (RFLP) using the restriction enzymes HinfI and MspI and were differentiated by culture on CHROMagar Candida medium. Results:The frequency of Candida spp. was higher in diabetic patients compared to non-diabetics. The most frequent Candida spp. in the diabetic patients were Candida albicans (%36.2), C. Krusei (%10.4), C. Glabrata (%5.1), and C. tropcalis .(%3.4)Likewise, C. albicans was the most frequent species (%27) in the non-diabetic individuals. In this study, the results of both methods for identification of the isolates were consistent with each other.Conclusion:Xerostomia and disturbance of physiological factors including pH and glucose can promote overgrowth of Candida flora in the oral cavity. These factors are considered important predisposing factors for oral candidiasis in diabetic patients. In the present study, it was observed that application of CHROMagar Candida and PCR-RFLP methods at the same time contributes to more accurate identification of isolates.

Polymorphism in exon 2 encoding the putative ligand binding pocket of the bovine insulin-like growth factor 1 receptor affects milk traits in four different cattle breeds.

As a member of the somatotropic axis, insulin-like growth factor I receptor (IGF1R) seems to be a promising candidate gene. Two silent polymorphisms, identified by MspI and TaqI restriction enzymes, were selected within exon 2, encoding the majority of the putative ligand binding pocket. A total of 1169 cows of four pure breeds (Polish Holstein Friesian, Montbeliarde, Jersey and Holstein Friesian) were genotyped. The T (IGF1R/e2/MspI) and G (IGF1R/e2/TaqI) alleles were found to be prevalent. Three combinations of genotypes (TT/GG, TT/AG and CT/GG) were associated with the highest productivity (milk, protein and fat yields) among all breeds under study, as opposed to individuals carrying the worst CC/AA combination. In view of the specific structure of the ligand binding pocket and the significance of insulin-like growth factor I signalling promoting the development and differentiation in a variety of tissues (not only limited to mammary gland), the existence of missense mutation is unlikely. Potential mutations are likely limited to mRNA transcription and further post-transcriptional modifications. Further investigations should follow searching for the most useful IGF1R haplotypes, associated with higher milk production traits, exerting at the same time positive or neutral impact on health and welfare of individuals.

Renalase Gene rs2576178 Polymorphism in Hemodialysis Patients: Study in Bosnia and Herzegovina

Introduction:Renalase is a protein secreted in kidneys and considered as a blood pressure modulator. High rates of hypertension and its regulation in patients on hemodialysis demands search for potential cause and treatment. The aim of this study was to determine the genotype and allele frequencies of renalase gene rs2576178 polymorphism in population from Bosnia and Herzegovina. Also, the objective of present study was to find the possible association between renalase gene rs2576178 polymorphism and hypertension in patients on hemodialysis.Material and Methods:The genotype of renalase gene rs2576178 polymorphism was determined in 137 participants (100 patients on hemodialysis and 37 controls), using polymerase chain reaction (PCR) and subsequent cleavage with MspI restriction endonuclease. Genotype and allele frequencies were assessed for Hardy-Weinberg equilibrium using a Chi-squared test. The value of P<0.05 was considered as statistically significant.Results:Comparison of genotype distribution and allele frequency in participants on hemodialysis with and without hypertension, and healthy control showed no statistical difference.Conclusion:The results of the study suggest that renalase gene rs2576178 polymorphism is not a factor that influences blood pressure in patients on hemodialysis.

제주도에 서식하는 Petrosia corticata 해면의 배양가능한 공생세균 군집구조의 계절적 차이

The community structure of cultivable bacteria associated with the marine sponge, Petrosia corticata, collected from Jeju Island in summer (September) of 2012 and winter (January) of 2013, were compared by the PCR-ARDRA method. Bacterial strains were cultured for 4 days at <TEX>$26^{\circ}C$</TEX> on Zobell medium and marine agar medium. After PCR amplification of 16S rRNA gene of individual strains, the restriction enzymes MspI and HaeIII were used to make restriction patterns. As a result, 24 ARDRA patterns from the summer sponge and 20 ARDRA patterns from the winter sponge were obtained. The sequencing result of 1-3 selected strains from each pattern showed over 98% similarities with the known sequences from the public database. At the phylum level, the bacterial community structures of both sponges (summer and winter) were identical qualitatively and composed of 4 phyla : Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Alphaproteobacteria accounted for 42.5% of total in summer sponge and 25.2% in winter, decreasing in the winter sample. Gammaproteobacteria accounted for 27.5% of total in summer sponge and 35.2% in winter, increasing in the winter sample. At the genus and species level, summer sponge had more diverse bacterial communities than winter sponge. Actinobacteria, Bacteroidetes, and Firmicutes increased in the winter sample.

ARDRA와 DGGE를 이용한 Halichondria panicea 해면의 공생세균 다양성

Department of Biological Science and Biotechnology, Hannam University, Daejeon 34054, Republic of Korea(Received December 23, 2015; Accepted December 24, 2015)ABSTRACT: Culture-dependent ARDRA and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Halichondria panicea collected from Jeju Island. A total of 120 bacterial strains associated with the sponge were cultivated using modified Zobell and Marine agar media. PCR amplicons of the 16S rRNA gene from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rRNA gene sequences derived from ARDRA patterns showed more than 96% similarities compared with known bacterial species, and the isolates belonged to four classes, Alphaproteobacteria , Gammaproteobacteria , Bacteroidetes , and Firmicutes , of which Alphaproteobacteria was dominant. DGGE fingerprinting of 16S rRNA genes amplified from the sponge-derived total gDNA showed 14 DGGE bands, and their sequences showed 100% similarities compared with the sequences available in GenBank. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of seven classes, including Alphaproteobacteria , Gammaproteobacteria , Acidobacteria , Actinobacteira , Bacteroidetes , Cyanobacteria , and Chloroflexi . According to both the ARDRA and DGGE methods, three classes, Alphaproteobacteria , Gamma-proteobacteria , and Bacteroidetes , were commonly found in H. panicea. However, overall bacterial community in the sponge differed depending on the analysis methods. Sponge showed more various bacterial community structures in culture independent method than in culture-dependent method.Key words: Halichondria panicea , 16S rRNA gene, ARDRA, bacterial diversity, DGGE, marine sponge

Molecular diversity and multifarious plant growth promoting attributes of Bacilli associated with wheat (Triticum aestivum L.) rhizosphere from six diverse agro-ecological zones of India.

The diversity of culturable Bacilli was investigated in six wheat cultivating agro-ecological zones of India viz: northern hills, north western plains, north eastern plains, central, peninsular, and southern hills. These agro-ecological regions are based on the climatic conditions such as pH, salinity, drought, and temperature. A total of 395 Bacilli were isolated by heat enrichment and different growth media. Amplified ribosomal DNA restriction analysis using three restriction enzymes AluI, MspI, and HaeIII led to the clustering of these isolates into 19-27 clusters in the different zones at >70% similarity index, adding up to 137 groups. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 55 distinct Bacilli that could be grouped in five families, Bacillaceae (68%), Paenibacillaceae (15%), Planococcaceae (8%), Staphylococcaceae (7%), and Bacillales incertae sedis (2%), which included eight genera namely Bacillus, Exiguobacterium, Lysinibacillus, Paenibacillus, Planococcus, Planomicrobium, Sporosarcina, and Staphylococcus. All 395 isolated Bacilli were screened for their plant growth promoting attributes, which included direct-plant growth promoting (solubilization of phosphorus, potassium, and zinc; production of phytohormones; 1-aminocyclopropane-1-carboxylate deaminase activity and nitrogen fixation), and indirect-plant growth promotion (antagonistic, production of lytic enzymes, siderophore, hydrogen cyanide, and ammonia). To our knowledge, this is the first report for the presence of Bacillus endophyticus, Paenibacillus xylanexedens, Planococcus citreus, Planomicrobium okeanokoites, Sporosarcina sp., and Staphylococcus succinus in wheat rhizosphere and exhibit multifunctional PGP attributes. These niche-specific and multifarious PGP Bacilli may serve as inoculants for crops growing in respective climatic conditions.

Single nucleotide polymorphisms of APOA1 gene and their relationship with serum apolipoprotein A-I concentrations in the native population of Assam

BackgroundThere is a growing interest in the role of allelic variants of the APOA1 gene in relation to a number of disorders. We described two common polymorphisms of the APOA1 gene, G-75A and C+83T and investigated their potential influence on the serum apolipoprotein A-I (apo A-I) levels in the native population of Assam — a region that is ethnically distinct and from where no information is hitherto available. MethodsBlood samples were collected from 150 healthy volunteers. Apo A-I levels were estimated by immunoturbidometry. Genotyping was done by a PCR-RFLP method that involved DNA extraction from whole blood, followed by polymerase chain reaction and digestion of the PCR product by MspI restriction enzyme, and analysis of fragment sizes in 12% polyacrylamide gel. ResultsThe GG variant at G-75A locus and CC variant at C+83T locus were the most prevalent. GG/CC was the most common combination. Homozygous TT genotype was not detected in any of the subjects. The rare allele frequencies for the G-75A and C+83T sites were found to be 0.22 and 0.06 respectively, which significantly differed from those reported in some other populations in neighbouring regions. Serum apo A-I concentrations did not vary significantly across the detected genotypes. These findings were consistent in both sexes. ConclusionWe described the distribution of the G-75A and C+83T polymorphisms of the APOA1 gene in the population of Assam for the first time. These polymorphisms were not found to directly influence apo A-I concentrations in this population either individually or synergistically.

The use of multiple restriction enzymes in terminal restriction fragment length polymorphism analysis and identification of performance-related caecal bacterial groups in growing broiler chickens

SUMMARYFour restriction enzymes (AluI, HhaI, MspI and RsaI), either individually or in combination, were used in terminal restriction fragment length polymorphism (T-RFLP) analysis to: (i) characterize the chicken intestinal bacterial community; and (ii) tentatively identify intestinal bacterial groups related with increased performance parameters in broiler chickens. Balanced commercial diets free of any feed antibiotics were offered to broilers assigned randomly to one of the five dietary treatments: control (C) (commercial diet with no additive), inulin (I), fructose caramel, propyl propane thiosulphonate (PTS-O)-45 and PTS-O-90. Except for the inulin-supplemented diet, multivariate statistical analysis of T-RFLP profiles based on individual enzymes or their combinations showed that the caecal bacterial community composition was significantly different among diets, particularly between the control and the supplemented diets. Individual RsaI and the combination AluI + RsaI proved to be the most useful to discriminate between dietary treatments. Clostridiaceae 1, Lachnospiraceae, Ruminococcaceae and Micrococcaceae were tentatively identified as those families most likely to be implicated in defining the caecal microbiota composition of growing broiler chickens, and also as those most closely related with differences in productive parameters.

Polimorfismo A192G de INHA y su asociación con características lecheras en ganado Holstein de Antioquia

El objetivo de esta investigación fue determinar el efecto del polimorfismo A192G en algunos parámetros productivos y reproductivos en vacas Holstein de Antioquia. Para esto, se utilizó la técnica PCR-RFLP para amplificar un segmento de 249 pb del gen de inhibina alfa bovina (INHA) el cual fue digerido con la enzima de restricción MspI. El efecto de los genotipos de INHA en las características productivas y reproductivas se determinó con un Modelo Linear Mixto y la prueba de Tukey se usó para establecer diferencias significativas entre las medias de los tres genotipos. El efecto de sustitución alélica se estudió a través de un modelo de regresión lineal donde los genotipos AA, AG y GG fueron transformados a una escala cuantitativa de 0, 1 y 2, respectivamente de acuerdo al número de alelos G poseídos. En el ganado Holstein de Antioquia el genotipo más común fue el AG (0,529) seguido por el AA (0,269) y el genotipo GG (0,201). Las frecuencias alélicas fueron 0,534 para A y 0.466 para G. Los genotipos de INHA no ejercieron un efecto significativo en los principales parámetros reproductivos, excepto para porcentaje de grasa donde los animales GG presentaron el mayor valor para esta característica. A pesar de que no hubo un efecto significativo de este polimorfismo en los parámetros reproductivos, la prueba de Tukey mostró que el alelo G tiende a tener un efecto negativo en la fertilidad de las hembras, aumentando el intervalo entre partos y el número de servicios por concepción, disminuyendo la eficiencia reproductiva de los hatos. Se requieren más estudios para determinar si estos SNP pueden ser utilizados como marcadores moleculares.

Use of restriction fragment length polymorphism to identify Candida species, related to onychomycosis.

Background:Onychomycosis is one of the most common clinical forms of fungal infections due to both filamentous fungi and yeasts. The genus of Candida is one of the most prominent causes of onychomycosis in all around the world. Although Candida albicans is still the most frequent cause of nail infections, use of broad-spectrum antifungal agents has led to a shift in the etiology of C. albicans to non-albicans species. The aim of the present study is rapid and precise identification of candida species isolated from nail infection by using of PCR-RFLP technique.Materials and Methods:A total of 360 clinical yeast strains were collected from nail infections in Iran. Genomic DNA was extracted using FTA; cards. ITS1-5.8SrDNA-ITS2 region was amplified using universal primers and subsequently products were digested with the restriction enzyme MspI. For identification of newly described species (C. parapsilosis complex), the SADH gene was amplified, followed by digestion with Nla III restriction enzyme.Results:Candida albicans was the most commonly isolated species (41.1%), followed by C. parapsilosis (21.4%), C. tropicalis (12.8%), C. kefyr (9.4%), C. krusei (5.5%), C. orthopsilosis (4.1%), C. glabrata (2.8%), C. guilliermondii (1.4%), C. rugosa (0.8%), and C. lusitaniae (0.5%). Patients in the age groups of 51-60 and 81-90 years had the highest and lowest distribution of positive specimens, respectively.Conclusion:Rapid and precise identification of Candida species from clinical specimens lead to appropriate therapeutic plans.

Identification of polymorphisms of XRCC1 gene in patients with cancer in a city of northern Brazil

Introduction: Cancer is considered a genetic disease. For this reason, identification and characterization of the genes involved in its origin and progression are of fundamental importance in understanding its molecular basis. Objective: Our objective was to determine whether people from Macapa with a diagnosis of cancer have genetic polymorphisms related to the XRCC1 gene. Materials and methods: We analyzed 30 samples of deoxyribonucleic acid (DNA) of cases with cancer and 30 control samples. All samples were amplified and analyzed by the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method, with the use of restriction enzyme MspI. Results: Regarding the 194T polymorphism, we found that all samples of the cases presented the polymorphic allele Trp (Arg/Trp). In control samples, 96.6% also identify the polymorphic allele Trp and, among these, one was homozygous for the same allele (Trp/Trp). Regarding the 399A polymorphism, 83.3% of the cases and 23.3% of the controls had the Arg/ Gln genotype, respectively. We found that 73.3% of controls and 16.6% of cases had the Arg/Arg genotype. Among the controls, we found only a sample that was homozygous for the polymorphic allele Trp/Trp. Conclusion: Our results demonstrated the allele frequency of 194Trp polymorphism in both sample groups analyzed. We also found a significant number of polymorphic allele 399A in people with cancer. Thus, we can highlight 399Gln polymorphism as a genetic marker of cancer risk in this population.

The diversity of non-methanogenic bacteria involved in biogas production from tofu-processing wastewater

Sunarto, Tsaaqifah H, Purwoko C, Pangastuti A. 2014. The diversity of non-methanogenic bacteria involved in biogasproduction from tofu-processing wastewater. Biodiversitas 16: 62-68. The energy crisis is an important current issue, so diversificationof energy is needed to solve the problem. Biogas from tofu-processing wastewater is one potential alternative energy to be developedbecause it has high organic matter content. The amount of methane formed by methanogens is also influenced by the presence ofbacteria, so it is necessary to do research on the diversity of non-methanogenic bacterial communities. This study aimed to determine thediversity of non-methanogenic bacteria in anaerobic fermentation using ARDRA (Amplified Ribosomal DNA Restriction Analysis)method. Tofu-processing wastewater was anaerobically fermented, followed by DNA extraction, amplification of 16S rRNA markergene, and cloning. Then, restriction was done using MspI and AluI restriction enzymes, followed by sequencing of different patterns.The data were analyzed to know the diversity of non-methanogenic bacteria and the relative importance of each phylotype in thecommunity using Shannon-Wiener index of diversity (H’) and evenness (E).The results showed that there were 38 positive clones of16S rRNA genes clones. The sequencing using BLASTn analysis showed the presence of 10 species of non-methanogenic bacteria.There were four sequences with 97% similarity, indicating that the bacteria belong to species from GeneBank data. There were 6sequences that had 96% similarity with the closest relative in the database, indicating that these bacteria are new species. The Shanon-Wiener index was 1.291, categorized as low and the evenness was 0.561, categorized as low because there was dominance of a species.

Association of an Exon SNP of SLC2A9 Gene with Hyperuricemia Complicated with Type 2 Diabetes Mellitus in the Chinese Male Han Population.

Several recent genome-wide association studies and following studies have identified that genetic variants of SLC2A9 are associated with hyperuricemia (HUA) and diabetes mellitus (DM). Here, we set to investigate whether the exon 9 of SLC2A9 gene variations is associated with HUA complicated with Type 2 DM (T2DM) in the Chinese male Han population. The present study was designed to study rs2280205 polymorphism in exon 9 of SLC2A9 in 232 Chinese male subjects. Rs2280205 locus was genotyped in 52 T2DM subjects, 65 HUA subjects, 55 subjects with HUA complicated with T2DM, as well as 60 control subjects in this study. DNA from peripheral blood was purified and amplified by polymerase chain reaction (PCR). The PCR products were then digested by restriction enzyme MSPI, and part of PCR products was sequenced and analyzed. There was no significant difference in the levels of cholesterol, creatinine, and urea nitrogen between the Control Group and the HUA group. There was also no significant difference in levels of cholesterol between the DM group and Control Group. No significant difference in cholesterol and uric acid was observed between the HUA group and the HUA accompanied with DM group (P > 0.05). However, there was no statistical significance in the genotype frequency in these groups (P > 0.01). Results of the present study suggest that the exon 9 of SLC2A9 gene 109C/T polymorphism is not associated with HUA and diabetes in population living in the coastal area of Shandong province, China.

Leptin receptor polymorphism Gln223Arg (rs1137101) in oral squamous cell carcinoma and potentially malignant oral lesions.

The purpose of this study was to assess the LEPR gene Gln223Arg polymorphism (rs1137101) in oral squamous cell carcinoma (OSCC) and in potentially malignant oral lesions (PMOL) in comparison to normal oral mucosa in a Brazilian population. Smokers (n = 89) were selected from a representative sample of 471 individuals from the general population of Montes Claros, Brazil. Participants were age and gender matched to patients with OSCC (n = 25) and oral epithelial dysplasia (n = 25). We investigated the LEPR Gln223Arg polymorphism (A>G; rs1137101) in these groups. Genotype variants were assessed by RFLP-PCR, using MspI (HPAII) restriction endonuclease. The institutional review board of the Universidade Estadual de Montes Claros approved the study (process number 2667/2011). Written informed consent for this study was obtained from all participants. The GG genotype (Arg223Arg) appears to be the more relevant polymorphic variant in OSCC. It occurred, approximately, twice as frequently in OSCC patients than in the general population. In contrast, the A allele in its homozygosis form (Gln223Gln) is significantly associated with the development of PMOL; 80% of the samples from the PMOL group exhibit AA genotype. Our findings suggest new insights regarding LEPR gene variations in the development of OSCC and PMOL.

Phytophthora Taxon Pgchlamydo is a Cause of Shoot Blight and Root and Collar Rot of Viburnum tinus in Italy.

The quarantine pathogen Phytophthora ramorum has recently been found on dying Viburnum tinus in the nursery area of Pistoia, central Italy (43°56'0″ N, 11°1'0″ E) (3). As part of a surveillance program aimed at detecting P. ramorum in this area, the Phytophthora taxon Pgchlamydo was consistently found associated with symptomatic V. tinus. The crowns of these plants were wilted, and some plants also showed root and collar rot and underbark necrosis. Water courses adjacent to the nursery with the infected V. tinus were tested for the pathogen. Samples from seven symptomatic plants were placed on a selective V8A-PARPNH medium within 24 h from sampling. Tissue pieces (2 mm2) of 12 baits (apple fruits) exposed for a week in water bodies were plated on the same medium. Cottony colonies arose after 2 to 3 days of incubation at 23°C in the dark and were transferred to potato dextrose agar (PDA) in purity. Mycelial DNA was extracted with a commercial kit (Sigma-Aldrich). The rDNA ITS region and a portion of the mtDNA cox1 gene were PCR-amplified and the amplicons digested with the restriction enzymes MspI and AluI (for the ITS region) and RsaI (for the cox1 gene region). Isolates R7 from V. tinus, and ES2M5, ES2M11, and ES1M12 from the water bodies belonged to the same taxon based on restriction analysis of both DNA regions coupled with ITS-rDNA sequence homology (GenBank Accession Nos. KJ396773 to 76). A BLAST search in GenBank found that all isolates had a 99% identity in the ITS-rDNA with the Phytophthora ITS Clade 6 member P. taxon Pgchlamydo. Sporangia produced after incubation in filtered pond water for 24 h were mostly ovoid (sometimes obpyriform), non-papillate, non-caducous. Some sporangia were emptied with external proliferation and had hyphal swellings. Thirty sporangia were measured and averaged 42.4 ± 6.2 × 29.9 ± 3.5 μm (range 30.0 to 56.1 × 22.5 to 38.0), with a length/width ratio of 1.4 ± 0.2 (1.2 to 2.0), and exit pores of 11.7 ± 1.5 μm (9.0 to 14.6). Optimum colony growth on V8A at 30°C was 4.4 ± 0.4 mm day-1, and the maximum temperature for growth was 32°C. Inoculation on twigs of Fagus sylvatica and V. tinus produced necrotic lesions of 2.6 ± 0.5 cm (2.1 to 3.5) and 4.7± 0.5 cm (3.8 to 5.6) respectively after 3 weeks of incubation at 23°C in the dark. Inoculation on V. tinus leaves resulted in lesions averaging 3.3 ± 1.1 × 2.1 ± 0.6 cm (range 2 to 5 × 1.5 to 3) after 2 weeks of incubation at 23°C in the dark. Control plant material showed no symptoms.The Phytophthora taxon Pgchlamydo has been reported on several ornamental and woody species, including Arctostaphylos sp., Camellia spp., Laurus nobilis, Buxus sempervirens, Rhododendron sp., Arbutus unedo, Prunus sp., Pseudotsuga sp., and Sequoia sempervirens, in North America and Europe (1,2). This is the first report, to our knowledge, of this taxon on V. tinus in Italy. V. tinus is widely sold in European nurseries, and it is also one of the most common hosts of P. ramorum (4). The fact that V. tinus is a host for both oomycetes, and the two microorganisms induce a similar symptomology (wilt), might complicate the control efforts of the phytosanitary inspection services aimed at restricting P. ramorum foci in Europe.

Abstract 396: TET oxidase activity accumulated on methyl-CpG sites extensively upregulates methylated genes through DNA demethylation

Abstract Ten-Eleven Translocation (TET) proteins play an important role in regulating DNA methylation fidelity and their inactivation contributes to the DNA hypermethylation phenotype in cancer; they are Fe²+- and 2-oxoglutarate-dependent dioxygenases that successively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) and these oxidized methylcytosines are important intermediates in the process of 5mC demethylation. TET proteins convert 5mC to 5hmC in vitro, but it is difficult to explore the process of DNA demethylation and their effects on endogenous hypermethylated genes in cells. In this study, we tried to develop a system that accumulates TET oxidase activity at hypermethylated promoters. To accomplish this, we constructed a fusion protein-producing vector consisting of methyl-CpG binding domain (MBD) and TET1 catalytic domain (TET1 CD) and asked whether this fusion protein (MBD-TET1 CD) could lead to DNA demethylation and gene reactivation. We first prepared DNA constructs that contain wild-type (wt) TET1 CD as well as catalytically inactive TET1 CD mutant (mut) with or without MBD from MBD2 protein and then transfected each construct into human embryonic kidney cell line 293T. We isolated stably transfected 293T cell lines and analyzed the expression of genes transcriptionally silenced by DNA hypermethylation using RT-PCR. Surprisingly, only plasmids containing TET1 CDwt reactivated silenced MAL, SOX17, TRH, MT1M, and MLH1 genes in a MBD dependent manner. To clarify the mechanisms of the antisilencing activities in clones stably expressed MBD-TET1 CDwt, we examined the CpG islands in MAL, TRH, and MT1M promoters by genomic sequencing after bisulfite modifications. Almost all CpG sites were methylated in parental cell line 293T, a TET1 CDwt clone, a TET1 CDmut clone, and a MBD-TET1 CDmut clone. On the other hand, MBD-TET1 CDwt clones showed an extensive demethylation at CpG islands of all three genes. In addition, because bisulfite sequencing does not distinguish between 5mC and 5hmC, we converted 5hmC to glucosylated form and discriminated between 5mC and 5hmC using restriction enzymes MspI and HpaII. Quantitative real time PCR analysis clearly demonstrated that MBD-TET1 CDwt promoted demethylation at CpG sites with some 5hmC bases. We further examined the changes in global gene expression in stable cell lines containing different TET constructs by microarray analyses. All top 50 genes upregulated by MBD-TET1 CDwt-stably expressing cell lines contained CpG islands and interestingly, 43 of top 50 genes were also upregulated after treatment with DNA demethylating agent, 5-azacytidine. These results suggest that specific recruitment of TET oxidase activity on hypermethylated gene promoters induces DNA demethylation and promotes reactivation of epigenetically silenced genes. Citation Format: Shinichi Fukushige, Yasuhiko Mizuguchi, Akira Horii. TET oxidase activity accumulated on methyl-CpG sites extensively upregulates methylated genes through DNA demethylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 396. doi:10.1158/1538-7445.AM2014-396