Restriction Enzymes HinfI Research Articles

Distribution of Candida in the oral cavity and its differentiation based on the internally transcribed spacer (ITS) regions of rDNA

This study aimed to determine the distribution of Candida species in the oral cavity and differentiate the species based on PCR amplification, including HinfI and MspI digestion, in order to assess the effectiveness of using the rDNA region for species identification. Samples from saliva as well as palate, tongue and cheek mucosa surfaces were collected from 45 individuals, consisting of three groups: periodontal disease patients; denture-wearers; and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for colony-forming units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction, based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI-5.8S-ITSII PCR product was digested by HinfI and MspI restriction enzymes. The microbial loads on all sites of the denture-wearers were found to be significantly higher than control, while in the periodontal disease group only the microbial loads on the tongue were significantly higher than control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to those of seven control species, allowing the differentiation of all seven species and the identification of 14 species from the clinical samples. The MspI restriction digest was not able to distinguish between C. albicans and C. dubliniensis, whereas the HinfI digest could not distinguish between C. tropicalis and C. parapsilosis. It was concluded that PCR-RFLP of the candidal rDNA region has potential for species identification. This study demonstrates the potential use of candidal rDNA as a means for identifying Candida species, based on genotype. The results also indicate the possibility of constructing genetic probes that target specific restriction fragments in the ITSI-5.8S-ITSII region, enabling swift and precise identification of Candida species.

PCR-RFLP Based Method for Molecular Differentiation of Sand Fly Species <I>Phlebotomus argentipes</I>, <I>Phlebotomus papatasi</I>, and <I>Sergentomyia babu</I> Found in India

PCR-Restriction fragment length polymorphism is a time saving and accurate technique to differentiate closely related organisms. In the regions endemic for visceral leishmaniasis in India, various species of morphological similar sand fly exist but only female Phlebotomus argentipes is the vector for visceral leishmaniasis. In the current study primers were designed targeting the 18S rRNA encoding gene that showed amplification in all the major sand fly species found in India. The amplified fragments were further digested using the HinfI or HpaII restriction enzymes. Each of the restriction enzyme produced a species specific restriction patterns, which can easily be used to identify specific sand fly species. This technique can be used in the identification of sand fly species.

Chloroplast Genome Diversity in Portuguese Grapevine (Vitis vinifera L.) Cultivars

Grapevine chloroplast (cp) DNA diversity was analysed for the first time through amplification and digestion of fragments of the large single copy (LSC) region by polymerase chain reaction-restriction fragment length polymorphism methodology and also by amplification of three microsatellite loci, previously described as polymorphic in grapevine. Thirty-eight grapevine cultivars collected mainly in the North of Portugal, including some neglected cultivars, four international cultivars (Chasselas, Muscat of Alexandria, Muscat of Hamburg and Pinot) and Vitis riparia and Vitis rupestris, were used in this study with the main goal of finding out their cpDNA diversity and compare the obtained results with previously published data on cultivars from other regions to ascertain their possible origin. Two different alleles were found in each of the three cpSSR loci. Allele variants of the three loci combined in a total of three different haplotypes (A, B and D). The most frequent haplotype, A, was previously reported as the most frequent in Iberian Peninsula and Occidental Europe. Haplotype B was unique to Rabigato, Muscat of Alexandria, V. riparia and V. rupestris. This haplotype was previously proposed to be an ancestral haplotype. Twenty-seven fragments of the LSC region of Vitis vinifera cpDNA were amplified and then digested with HinfI and TaqI restriction enzymes. Polymorphisms were found in the trnT-psbC (TC) and orf184-petA (OA) fragments. In the TC fragment, the polymorphism corresponds to a point mutation in a restriction site of TaqI and is only present in all cultivars with cpSSR haplotype D. In the OA fragment, a short deletion exclusive to the Rabigato cultivar was found. In this case, one sequence tagged site-based marker was developed and will be very useful in future phylogenetic and fingerprinting studies in a broader number of cultivars and in wild grapevine populations. Inference about the progenitors of the Touriga Franca cultivar is done. The present work supports and completes its origin as a descendent of the female and male parents, Marufo and Touriga Nacional.

Restriction analysis of an amplified rodA gene fragment to distinguish Aspergillus fumigatus var. ellipticus from Aspergillus fumigatus var. fumigatus

A previous multidisciplinary study indicated that gliotoxin-producing Aspergillus fumigatus Fresen. isolates from silage commodities mostly belonged to its variant A. fumigatus var. ellipticus Raper & Fennell. Sequence analysis revealed the presence of a single nucleotide polymorphism at five positions in a fragment of the rodA gene (coding for a hydrophobin rodletA protein) between Aspergillus fumigatus var. fumigatus and Aspergillus fumigatus var. ellipticus. A method was developed to distinguish these two types of isolates based on restriction analysis of this rodA gene fragment using the HinfI restriction enzyme. In addition, in silico analysis of 113 rodA gene fragments retrieved from GenBank was performed and confirmed the suitability of this method. In conclusion, the method developed in this study allows easy distinction between A. fumigatus var. fumigatus and its variant ellipticus. In combination with the earlier developed PCR-restriction fragment length polymorphism method of Staab et al. (2009, J Clin Microbiol 47: 2079), this method is part of a sequencing-independent identification scheme that allows for rapid distinction between similar species/variants within Aspergillus section Fumigati, specifically A. fumigatus, A. fumigatus var. ellipticus, Aspergillus lentulus Balajee & K.A. Marr, Neosartorya pseudofischeri S.W. Peterson and Neosartorya udagawae Y. Horie, Miyaji & Nishim.

P3.06 Comparison of High Resolution Melting and Restriction Fragment Length Polymorphism to Genotype Snp in Formalin Fixed Paraffin-Embedded Breast Cancer Tissue

ABSTRACT SNPs of NQO1, p53, and MDM2 have been correlated with pathological response or overall survival of breast cancer patients treated with anthracycline based chemotherapy. Therefore, the ability to detect these SNPs may be useful for treatment decision. In this study, we genotyped FFPE breast cancer samples to detect those SNP. Objective The aim of this study was to know the accuracy of HRM (High Resolution Melting) method to genotype SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G's in comparison to RFLP (Restriction Fragment Length Polymorphism) from archived formalin fixed paraffin embedded (FFPE) breast cancer blocks. Materials and Methods FFPE blocks were collected from anatomic pathology laboratory. DNA were isolated, and PCR amplified followed by either RFLP or HRM methods. Restriction enzymes HinfI, BstUI, and MspA1I were used in RFLP to genotype NQO1, p53, and MDM2 SNPs, respectively. The primer design which been used was the same between HRM and RFLP. The results of HRM were analyzed using melting, normalization, and differentiation plot curves. Sequencing technique has been used to resolve discrepancy between RFLP and HRM results. Results We screened SNPs on breast cancer FFPE samples using HRM and RFLP. The rates of genotyping agreement between both methods are 69, 23% (9/13 samples), 80% (8/10 samples), and 100% (7/7 samples) for NQO1, p53, and MDM2 SNPs, respectively. Conclusion HRM is a faster method than RFLP to genotype important breast cancer SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G. Nevertheless, RFLP is more reliable method than HRM when the used primers sets are not optimized for HRM.

A Molecular Key for the Identification of Blow Flies in Southeastern Nebraska*

Immature blow flies (Calliphoridae) are typically the first colonizers of cadavers. Identification of the early instars using traditional, morphology-based keys is difficult because of their small size, similarity, and simplicity in external morphology. Information derived from molecular genetic data would augment the accurate identification of immature flies. Nine species of blow flies commonly found in southeastern Nebraska were used to examine the utility of molecular-based keys. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) were investigated with 10 common, inexpensive, restriction enzymes from an amplicon of approximately 1500 bp spanning the mitochondrial cytochrome oxidase I gene. A simple molecular taxonomic key, comprising RFLP from the restriction enzymes HinfI and DraI, enabled the differentiation of all species used. Further development of PCR-RFLP, including more extensive and intensive examination of blow flies, would benefit forensic laboratories in the accurate identification of evidence consisting of immature blow flies.

A PCR BASED RFLP SURVEY OF S-ALLELES IN WILD AND CULTIVATED ALMOND AND RELATED PRUNUS SPECIES

Almond is one of the most important nut crops in the world. The origin of almond is central and west Asia. About 30 wild species are identified in this region which represent a rich source of desirable characteristics useful in almond breeding programs. However, almonds express gametophytic self- and cross-incompatibility which affects breeding parent selection. Incompatibility in almond is controlled by a multi-allelic, single gene (S-locus). Recently chemical and molecular methods have been utilized to survey the S-alleles in almond cultivars and closely related species. The restriction fragment length polymorphism method using CAPs protocol was used to determin S-alleles in multiple accessions of wild almond and related Prunus species. Polymerase chain reactions were carried out using one set of degenerate primer pairs (EM-PC2consFD/EM-PC3consRD), and PCR products were digested by five restriction enzymes (TaqI, HinfI, DpnI, ApoI and AluI). The genotypes revealed different cutting paterns. ApoI and DpnI produced the most and the least bandings respectively. AluI, ApoI and HinfI digested the S7. ApoI was the only enzyme which could digest the S9. TaqI, ApoI and HinfI digested the S8 and S12.

Genotype distribution of methylenetetrahydrofolate reductase A1298C and C677T gene in Indonesian infertile men

Background: Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme of folate and methionin metabolism, making it crucial for DNA synthesis and methylation. Variants of MTHFR C677T and A1298C gene result in reduced plasma folate levels and increase the susceptibility to spermatogenic arrest. This research aims to analyses MTHFR C677T and A1298C gene polymorphism in Indonesian infertile men with azoospermia and oligozoospermia. Methods: This cross sectional study takes 3 mL of blood from 150 infertile men with oligozoospermia and azoospermia. MTHFR gene is analyzed using polymerase chain reaction technique (PCR) with specific primers. PCR-RFLP analysis of the MTHFR gene using restriction enzymes MboII and HinfI determines allotypes, both of SNP A1298C and C677T in oligozoospermia and azoospermia in Indonesian population. Results: The results show that the distribution of allotypes of MTHFR gene SNP A1298C and A677T is not significantly different (p>0.05) between patient groups with oligozoospermia and azoospermia. Conclusion: MTHFR gene polymorphisms, both of SNP A1298C and C677T are not associated with male infertility in Indonesian men including patients with severe oligozoospermia and azoospermia. (Med J Indones 2012;21:23-7) Keywords: DNA methylation, MTHFR, spermatogenic arrest

Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant

The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.

Analysis of TLR4 and TLR2 polymorphisms in inflammatory bowel disease in a Guangxi Zhuang population

To study the polymorphisms of toll-like receptor 4 (TLR4) gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp and susceptibility to inflammatory bowel disease (IBD) in the Zhuang population from Guangxi, China. A case-control study was performed from February 2007 to October 2011 which included 146 Zhuang patients with IBD in the experimental group and 164 healthy Zhuang subjects who acted as the control group. All patients and healthy subjects were from the Guangxi Zhuang Autonomous Region of China. Genomic DNA was extracted from intestinal tissue by the phenol chloroform method. TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp were amplified by polymerase chain reaction (PCR), and then detected by PCR-restriction fragment length polymorphism (RFLP). The TLR4 gene Asp299Gly was digested using Nco I restriction enzyme, and a single band of 249 bp was observed which showed that it was a wild type (AA). The TLR4 gene Thr399Ile was digested using Hinf Irestriction enzyme and only the wild type (CC) was detected. In addition, the TLR2 gene Arg677Trp was digested using Aci I restriction enzyme and only the wild type (CC) was detected. The TLR2 gene Arg753Gln was digested using Pst I restriction enzyme. Only the wild type (GG) as a single band of 254 bp was observed during RFLP. Overall, no heterozygous or homozygous single nucleotide polymorphism mutations were found in patients with Crohn's disease and ulcerative colitis both in the TLR4 gene Asp299Gly, Thr399Ile and the TLR2 gene Arg677Trp, Arg753Gln in the Zhuang population from the Guangxi Zhuang Autonomous Region of China. The TLR4 gene Asp299Gly, Thr399Ile and TLR2 gene Arg753Gln, Arg677Trp polymorphisms may not be associated with IBD in the Zhuang population from the Guangxi Zhuang Autonomous Region of China.

Genetic characterization of Pyrenophora graminea isolates and the reactions of some barley cultivars to leaf stripe disease under greenhouse conditions

The genetic diversity of Pyrenophora graminea, the causal agent of barley leaf stripe disease, was characterized by restriction fragment length polymorphism (RFLP) of the rDNA internal transcribed spacer region and intersimple sequence repeat (ISSR) analysis. A total of 45 P. graminea isolates recovered from different geographic origins and barley cultivars or genotypes produced identical RFLP banding patterns with HinfI, MspI, Hin6I and BsuRI restriction enzymes. ISSR analysis indicated that there was little genetic variability among the isolates. A dendrogram based on Jaccard's coefficient separated the isolates into 4 clusters with 12% dissimilarity. In the dendrogram, the isolates collected from different geographic regions in Turkey were clustered separately from the Italian isolates. These results indicated that the Turkish isolates of P. graminea were genetically homogeneous and may be derived from a single gene pool. In addition, 13 isolates were tested for pathogenic variability on 48 barley cultivars. The cultivars Durusu, Balkan 96 (Igri), Çumra 2001 and Anadolu 98 were found to be resistant to all leaf stripe isolates.

Improved Detection of the KIT D816V Mutation Using a Real-Time PCR Assay Allows a Finer Recognition of Patients with Indolent Systemic Mastocytosis

Abstract 5163The somatic D816V mutation of the KIT gene is present in the large majority of patients (>90%) with Systemic Mastocytosis (SM) and, therefore, is considered as one of the minor criteria for its diagnosis by the 2008 World Health Organization (WHO) classification. Patients with indolent SM (ISM) without skin involvement frequently have a low mast cell burden in the bone marrow so they do not satisfy the major criterion for diagnosis of SM (i.e. the presence of multifocal dense mast cell infiltrates in extracutaneous organs). A finer detection of clonality markers in mast cells through highly sensitive diagnostic tools is necessary for the correct individuation of SM patients.To improve detection of D816V KIT mutation, we tested bone marrow (BM) cells from 97 adult patients with a suspicion for SM referred to our Multidisciplinary Outpatient Clinic for Mastocytosis in Verona from May 2009 to June 2011 using both a traditional PCR/RFLP assay and a mismatch amplification real time PCR technique.All patients underwent to a BM evaluation with histology/cytology and flow cytometry, as described (Bonadonna et al, 2009). For D816V KIT mutation analyses, RNA was extracted on BM mononuclear cell fraction and split in two equal amounts for traditional PCR and real time PCR respectively. In the traditional technique a PCR product of 287 bp, corresponding to the second tyrosine kinase domain (TKDII), was digested with the restriction enzyme HinfI to detect the nucleotide change at codon 816, leading to D816V mutation, and screened by RFLP assay. Concurrently mutation detection was conducted by a real time PCR in combination with mismatched forward primer for D816V mutation (Amplification Refractory Mutation System, ARMS) and Taqman probe; also WT primer was used to control RNA quality and compare samples.According to the 2008 WHO criteria, a diagnosis of ISM was made in 73 out of 97 patients (38 males, median age 50 years). Forty-two patients (57.3%) did not present skin involvement. The major histological criterion was fulfilled in 24 patients (32.8%), while in the remaining patients the diagnosis was made for the presence of at least 3 minor criteria. The presence of the D816V mutation was detected by real time PCR in 100% of 73 patients with ISM as well as the presence of CD25+ aberrant mast cells by flow cytometry. All the other patients, not suffering from mastocytosis, were negative both at flow cytometry and real time PCR, so determining a 100% concordance between these two highly sensitive techniques. Conversely, traditional PCR assay could demonstrate D1816V mutation only in 47 patients (64%) with SM. These patients had significantly higher levels of serum tryptase (median 27.5 vs 19.5 ng/mL, p=0.04) and higher amount of CD25+ aberrant mast cells by flow cytometry (median percentage of mast cells on total mononuclear cells 0.06 vs 0.018, p<0.001) as compared to patients negative by traditional PCR assay. Among the 28 patients with serum tryptase <20 ng/mL, 13 (46.4%) did not display KIT mutation by traditional PCR and 11/13 did not meet the major criterion: applying a conventional PCR technique only, these patients would have not fulfilled at least three minor criteria, thus missing a right diagnosis.We suggest that a real time PCR technique for D816V KIT mutation analysis, along with a highly sensitive flow cytometry assay, could improve the recognition of patients with ISM, particularly those with a very low mast cell burden and absence of skin lesions. Disclosures:No relevant conflicts of interest to declare.

Species identification of the Northern shrimp ( Pandalus borealis) by polymerase chain reaction–restriction fragment length polymorphism and proteomic analysis

Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method was designed based on the amplification of the 16S rRNA/tRNA Val/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) and electrospray ionization–ion trap (ESI–IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results.

Genetic polymorphism of three genes associated with milk trait in Egyptian buffalo

In dairy farm animals, the principal goal of the selection is the improvement of milk yield and composition. The genes of milk proteins and hormones are excellent candidate genes for linkage analysis with quantitative trait loci (QTL) because of their biological significance on the quantitative traits of interest. Prolactin (PRL) is a polypeptide hormone with multiple functions, secreted mainly by the anterior pituitary gland. Prolactin’s biological activity consists of various roles in the reproduction, lactation and a number of homeostatic biological functions including immune functions. Casein proteins and their genetic variants have been reported as important factors associated with lactation performance, milk composition and cheese yield efficiency. Genetic variants of bovine kappa-casein (K-CN) gene are associated with milk protein content and have a significant influence on rennet clotting time, firmness and cheese yield of milk. The pituitary-specific transcription factor (PIT-1) gene is responsible for pituitary development and hormone secreting gene expression in mammals. PIT-1 is studied as a candidate genetic marker for growth, carcass and also for milk yield traits. Genomic DNA extracted from 100 healthy buffaloes was amplified using primers that were designed from the cattle PRL, K-CN and PIT-1 gene sequences. The amplified fragments of PRL (294-bp), K-CN (530-bp) and PIT-1 (451-bp) were digested with RsaI, HindIII and HinfI restriction enzymes, respectively. The results showed that all tested buffaloes are genotyped as GG for PRL, BB for K-CN and BB for PIT-1.

English

Sixty Malaysian CTV isolates were characterized by bi-directional polymerase chain reaction (BD-PCR) and restriction fragment length polymorphism (RFLP) analysis of their coat protein (CP) gene. In BD-PCR analysis, 392-bp fragments were amplified from seven isolates. The other 53 isolates produced only 320-bp fragments. RFLP patterns of RT-PCR products of CP gene digested with HinfI restriction enzyme were similar to I-IV, VI-VII and two new groups. Isolates AMK1, AMJ12, AMT38, AMT39 and AMT43 could not be classified when they were compared to any standard CTV digest pattern. These isolates produced a unique restriction pattern with two fragments of 210 and 300 bp and isolate AMI61 produced different restriction pattern with three fragments of about 100, 270 and 300 bp. Therefore these isolates were designated as Groups IX and X. These results suggest that CTV populations in Malaysia contain new genetic variants. Key words: Citrus tristeza virus (CTV), strains, coat protein (CP) gene, restriction fragment length polymorphism (RFLP), bi-directional PCR, Malaysia.

Methylenetetrahydrofolate Reductase Gene Polymorphisms in Egyptian Women with Unexplained Recurrent Pregnancy Loss

This work aims at testing for the association of the methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with unexplained recurrent pregnancy loss (RPL) among Egyptian women. Participants were 70 cases having a history of two or more events of unexplained RPL and 136 controls with a good obstetric history. Detection of MTHFR C677T and A1298C mutations was done by polymerase chain reaction with restriction fragment length polymorphisms assay using restriction enzymes HinfI and MboII respectively. Compared with controls, cases with unexplained pregnancy loss showed higher frequency of the homozygous mutant MTHFR 677 TT, 1298 CC genotypes, and the mutant haplotype 677T/1298C, although not reaching statistical significance. The frequency of 677 mutant genotypes (TT or TC) combined with either the mutant 1298 (CC or AC) or normal 1298 (AA) genotypes was significantly increased among cases with late-stage pregnancy loss versus those with early-stage pregnancy loss (p=0.001). There was also increased frequency of the 677 mutant genotypes among cases with secondary infertility compared with those with primary infertility and among cases with pregnancy loss >4 times compared with those with ≤4 times but with no statistical significance. Regarding other risk factors, it was noted that the frequency of mutations among cases with no or just one risk factor did not differ significantly from those having two or more risk factors (p=0.98). Mutations related to the MTHFR gene are increased but not statistically significant in Egyptian women with unexplained pregnancy loss. Interaction with other genetic variants might be speculated and need to be investigated.

Comparative cytogenetics and heterochromatic patterns in two species of the genus Acanthostracion (Ostraciidae: Tetraodontiformes)

Some groups of fish, such as those belonging to the Order Tetraodontiformes, may differ significantly in the amount and location of heterochromatin in the chromosomes. There is a marked variation in DNA content of more than seven-fold among the families of this Order. However, the karyoevolutionary mechanisms responsible for this variation are essentially unknown. The largest genomic contents are present in species of the family Ostraciidae (2.20–2.60 pg). The present study cytogenetically characterized two species of the family Ostraciidae, Acanthostracion polygonius and A. quadricornis, using conventional staining, C-bandings, Ag-NOR, CMA 3/DAPI, AluI, PstI, EcoRI, TaqI and HinfI restriction enzymes (REs) and double FISH with 18S and 5S rDNA probes. The karyotypes of both species showed 2n = 52 acrocentric chromosomes (FN = 52; chromosome arms) and pronounced conserved structural characteristics. A significant heterochromatic content was observed equilocally distributed in pericentromeric position in all the chromosome pairs. This condition is unusual in relation to the karyotypes of other families of Tetraodontiformes and probability is the cause of the higher DNA content in Ostraciidae. Given the role played by repetitive sequences in the genomic reorganization of this Order, it is suggested that the conspicuous heterochromatic blocks, present in the same chromosomal position and with apparently similar composition, may have arisen or undergo evolutionary changes in concert providing clues about the chromosomal mechanisms which led to extensive variation in genomic content of different Tetraodontiformes families.

Antimicrobial resistance in non-typhi Salmonella enterica isolated from humans and poultry in Palestine

The efficacy of chemotherapy can be compromised by drug resistance.This study was undertaken to describe the resistance profiles and fluoroquinolone resistance mechanism of non-typhoidal Salmonella (NTS) isolated from humans and poultry in West Bank, Palestine. One hundred and fifty-one isolates of NTS, obtained from humans (71) and poultry (80), collected between September 2005 and January 2007, were tested for susceptibility to ampicillin, gentamicin, tetracycline ceftriaxone, nalidixic acid and ciprofloxacin. Mutation patterns within gyrA were determined by direct sequencing or by digestion of PCR-amplified DNA fragments with the restriction enzyme HinfI. Resistance rates among human and poultry isolates were respectively 59% and 51% for ampicillin, 31% and 10% for gentamicin, 59% and 80% for tetracycline, 59% and 45% for nalidixic acid, and 30% and 15% for ciprofloxacin. All the isolates were susceptible to ceftriaxone. Mutations at positions 83 and/or 87 were detected in gyrA of isolates with resistance to nalidixic acid. Isolates which were resistant to nalidixic acid but susceptible to ciprofloxacin had a single gyr A gene mutation at point 87.This gene mutation was sufficient to induce a new phenotype (6 isolates) with decreased susceptibility to ciprofloxacin. Mutations in gyrA at positions 83 or 87 were the most prevalent mutation pattern of fluoroquinolone resistant NTS isolates but other unknown mechanisms are also present. Continued surveillance of antimicrobial resistance among NTS isolates is needed to mitigate the increasing prevalence of quinolone resistance.

Diversity of the desert truffleTerfezia boudieriChatin. in southern Tunisia

This study reports the genetic diversity of Terfezia boudieri collected from southern Tunisia. The study was carried out using 135 truffle fruiting bodies harvested from seven different locations. Twenty-eight Terfezia claveryi fruiting bodies were also sampled from one of the seven locations. A PCR-based technique was used to amplify the internal transcribed spacer (ITS) region of the rDNA, including the ITS1-5.8S-ITS2. The PCR products were digested with the four restriction enzymes RsaI, HhaI, AluI, and HinfI. Based on the HinfI patterns, T.boudieri specimens were separated into two different haplotypes (I and II). Nucleotide sequences of some representative amplicons were also obtained. Based on the phylogenetic results, three T.boudieri genotypes could be differentiated. One sequence, SKtb1, retrieved from PCR-RFLP of haplotype I, was obtained from a low pH soil in association with Helianthemum kahiricum . Based on the results presented in the current study, this isolate may represent a novel taxa within the Terfezia genus.

First Report of Heterodera ripae on Common Nettle in Spain.

Soil and root samples were collected from May to October 2010 from cultivated and wild plants during a survey for hop cyst nematodes. Cyst nematodes were detected in roots of common nettle (Urtica dioica L.) with severe plant yellowing in four natural areas of León and Burgos provinces, Spain, where common nettle is grown in organic farming systems as a substrate for pharmaceutical products. Cysts were isolated by flotation and sieving methods. Cysts and juveniles were analyzed by morphological and molecular methods. The cyst nematodes obtained from soil and plant samples from all four locations had uniform morphological and molecular characteristics that differed from those of Heterodera humuli. Cysts (n = 25) had the following characteristics: lemon shaped, yellow to pale brown; cyst wall with ridges forming an irregular zigzag pattern; young cysts covered by subcrystalline layer; vulval cone bifenestrate with circular or subcircular fenestrae; bullae absent; underbridge weak; body length (not including the neck) ranging from 295 to 489 μm (mean of 418 μm); body width ranging from 208 to 375 μm (mean of 310 μm); fenestrate length of 39 to 58 μm (mean of 46.5 μm) and width of 25.2 to 30.9 μm (mean of 25.1 μm); underbridge length from 51 to 90 μm (mean of 69.2 μm); and vulval slit length from 26 to 40 μm (mean of 33 μm). J2 (n = 20) had the following characteristics: body length ranging from 338 to 380 μm (mean of 359.3 μm); stylet length from 21 to 24 μm (mean of 22.1 μm) with knobs rather wide and slightly projecting anteriorly; tail conical with a length of 41 to 52.5 μm (mean of 45.6 μm) and hyaline part of tail ranging from 18 to 25 μm (mean of 23.3 μm); lateral field with four lines. All morphological data and characters were consistent with those of H. ripae (1). DNA from single cysts was extracted to amplify the internal transcribed spacer (ITS) region of rDNA by PCR with forward primer TW81 (5'-GTTTCCGTAGGTGAACCTGC-3') and reverse primer AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3') (2). The PCR product was digested by restriction enzymes (AluI, CfoI, HaeI, HinfI, PstI, RsaI, TaqI, and Tru9I) to obtain restriction fragment length polymorphism profiles (2). ITS products cloned and assayed using the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Salamanca, Spain) were subjected to a database search using BLAST (National Centre for Biotechnology Information) to confirm the identification. These sequences exhibited 99.0% similarity with that of a H. ripae isolate from Germany (GenBank Accession No. AF274407.1). In glasshouse proofs of pathogenicity with these populations of H. ripae, 25 full cysts placed in nylon net bags were inoculated in 9-cm-diameter pots with 10 replicates per plant. After 12 weeks, soil from each pot was dried and cysts extracted. Cysts did not develop on roots of common hop (Humulus lupulus L.) and hemp (Cannabis sativa L.), but in common nettle there was an increase in nematode populations, with all plants severely stunted and yellowing, which confirmed the nematodes' pathogenicity. H. ripae has been previously reported in Russia, Estonia, Latvia, Armenia, Moldova, Ukraine, Bulgaria, Slovakia, Germany, and Belgium (1). To our knowledge, this is the first report of H. ripae in Spain. The identification of H. ripae in nettle fields is important in this region where it could cause large yield reductions if not properly managed.