Restriction Enzymes HhaI Research Articles

First Report of New Phytoplasma Diseases Associated with Soybean, Sweet Pepper, and Passion Fruit in Costa Rica.

A new soybean disease outbreak occurred in 2002 in a soybean (Glycine max) plantation in Alajuela Province, Costa Rica. Symptoms on the affected plants included general stunting, small leaves, formation of excessive buds, and aborted seed pods. In the same region, two other diseases, one in sweet pepper (Capsicum annuum) fields and another affecting passion fruit (Passiflora edulis) vines, were also found. Symptoms on sweet pepper plants included unusually dark green leaves, some of which exhibited a rugose symptom with a zigzag pattern to the midvein, and purple vein discoloration. Passion fruit vines exhibited bud proliferation. Collectively, symptoms resembled those commonly attributed to phytoplasmal infections. Total nucleic acid was extracted from veinal tissues of leaves or buds (soybean). A nested PCR assay using primer pair P1/P7 followed by R16F2n/R16R2 (1) was employed for the detection of putative phytoplasmas that might be associated within symptomatic plants. All seven symptomatic plants (three soybean, three sweet pepper, and one passion fruit) tested, but not healthy controls, yielded positive results. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products using restriction enzymes AluI, BfaI, HhaI, MseI, and RsaI indicated that the three diseases were associated with a very similar or identical phytoplasma. RFLP patterns and sequence analysis of cloned 16S rDNAs (GenBank Accession Nos. FJ226068-FJ226073) revealed that the phytoplasma shared less than 97.5% sequence homology with all previously classified phytoplasmas, and, as such, represents a new taxon most closely related to 16SrXII group (1) strains. To our knowledge, this is the first report of a new phytoplasma associated with diseases of soybean, sweet pepper, and passion fruit in Costa Rica. Reference: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

Identification of Panulirus homarus Puerulus Larvae by Restriction Fragment Length Polymorphism of Mitochondrial Cytochrome Oxidase I Gene

Molecular identification of puerulus larvae of Panulirus homarus of the genus Panulirus from Indian coast was studied by employing Polymerase Chain Reaction, Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the mitochondrial DNA (mtDNA) Cytochrome Oxidase Gene (COI) by agarose gel electrophoresis and Denaturing Gradient Gel Electrophoresis (DGGE). The size of amplified fragment of COI gene was estimated to be approximately 1300 base pairs (bp). Single fragment amplification was recorded during different stages of the life cycle. The RFLP digestion was carried out using five different restriction enzymes (BsplI, HhaI, RsaI, TaqI and AluI). The RFLP profile of the different endonucleases, varied between 1-5 restriction types. RFLP analysis using endonuclease TaqI enabled identification of P. homarus during different stages of its life history.

Identification of CpG methylation of the SNRPN gene by methylation‐specific multiplex PCR

In this article, we show that methylation-specific multiplex PCR (MS-multiplex PCR) is a sensitive and specific single assay for detecting CpG methylation status as well as copy number aberrations. We used MS-multiplex PCR to simultaneously amplify three sequences: the 3' ends of the SNRPN gene (for unmethylated sequences), the KRITI gene (as internal control), and the promoter of the SNRPN gene containing CpG islands (for methylated sequences) after digestion with a methylation-sensitive restriction enzyme (HhaI). We established this duplex assay for the analysis of 38 individuals with Prader-Willi syndrome, 2 individuals with Angelman syndrome, and 28 unaffected individuals. By comparing the copy number of the three regions, the methylation status and the copy number changes can be easily distinguished by MS-multiplex PCR without the need of bisulfite treatment of the DNA. The data showed that MS-multiplex PCR allows for the estimation of the methylation level by comparing the copy number aberrations of unknown samples to the standards with a known methylated status. The in-house-designed MS-multiplex PCR protocol is a relatively simple, cost-effective, and highly reproducible approach as a significant strategy in clinical applications for epigenetics in a routine laboratory.

Identification of an isoschizomer of the HhaI DNA methyltransferase in Mycoplasma arthritidis

The genome of Mycoplasma arthritidis strain 158 has modified cytosine residues at AGCT sequences that render the DNA resistant to digestion with the AluI restriction endonuclease. The DNA methyltransferase responsible for the base modification has previously been designated MarI. From the complete genome sequence of M. arthritidis, we identify Marth_orf138 as a candidate marI gene. Marth_orf138 was cloned in Escherichia coli and its TGA codons converted to TGG. DNA isolated from E. coli cells expressing the modified Marth_orf138 gene was degraded by the AluI nuclease, indicating that Marth_orf138 does not code for MarI. However, the DNA from E. coli was found to have acquired resistance to the restriction endonuclease HhaI. Genomic DNA from M. arthritidis was also found to be resistant to HhaI (recognizes GCGC). The M. arthritidis isoschizomer of the HhaI DNA methyltransferase, coded by Marth_orf138, is designated MarII. Transformation of M. arthritidis was not significantly affected by modification of plasmid at HhaI sites, indicating that the mycoplasma lacks a restriction endonuclease that recognizes GCGC sites.

Capillary electropherograms for restriction fragment length polymorphism of Helicobacter Pylori

Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.

Bovine kappa-casein gene polymorphism and its association with milk production traits

Point mutations in exon IV of the bovine κ-casein (CSN3) gene determine two allelic variants, A and B. These variants were distinguished by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis in the indigenous Sahiwal and Tharparkar cattle breeds. DNA samples (252 Sahiwal and 56 Tharparkar) were analyzed for allelic variants of the CSN3 gene. Polymorphism was detected by digestion of PCR-amplified products with HindIII, HhaI and HaeIII restriction enzymes, followed by separation on 3% agarose gels, and resolved by ethidium bromide staining. Allele A of the κ-casein gene occurred at a higher frequency than allele B, in both Sahiwal and Tharparkar breeds. The genotypic frequencies of AA, AB, and BB in the Sahiwal and Tharparkar breeds were 0.758, 0.230 and 0.012, and 0.0.732, 0.250 and 0.018, respectively. The frequencies of alleles A and B in the Sahiwal and Tharparkar breeds were 0.873 and 0.127, and 0.857 and 0.143, respectively. Genotype BB of the kappa-casein gene had more influence on the monthly milk yield, 305-days milk yield, monthly solids-not-fat (SNF) yield, and monthly protein yield, in the Sahiwal cattle.

The application of asymmetric PCR-SSCP in gene mutation detecting

The advantages and disadvantages of asymmetric PCR-SSCP and the traditional PCR-SSCP were compared in this study. The mutations in 3′UTR of Smad4 gene of Luxi cattle and the Holstein cow were analyzed by asymmetric PCR-SSCP and one insert “T” mutation and one G/A mutation in this region were found. The G/A mutation created a HhaI restriction enzyme digestion position and the frequencies studied by asymmetric PCR-SSCP and HhaI-RFLP in 116 Luxi cattle and 75 Holstein cows were all the same. The asymmetric PCR-SSCP had fewer, clearer and more stabile bands than traditional PCR-SSCP. This indicates that the asymmetric PCR-SSCP is suited for mutation detection.

Parentally imprinted allele (PIA) typing in the differentially methylated region upstream of the human H19 gene

The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study, the H19FR1 and H19FR2 haplotype polymorphisms including four and three SNPs, respectively, upstream of the H19 gene according to the GenBank sequence (accession no. AF125183) were investigated. Five haplotypes and nine genotypes were detected for H19FR1 in the Chinese Han population by means of PCR and subsequent denaturing gradient gel electrophoresis (DGGE). The power of discrimination (Dp), polymorphism information content (PIC) and probability of paternity exclusion (PE) were estimated to be 0.803, 0.58 and 0.322, respectively. For the H19FR2, two haplotypes and three genotyes were observed, and the Dp, PIC and PE were 0.626, 0.37 and 0.162, respectively. Sequencing results showed that only two of the four reported SNPs, a7342g and g7547a, were detected in H19FR1 in the Chinese Han population, and two new SNPs, g7351c and a7357g, were found. In the H19FR2 region, only one of the three reported SNPs, a8097g, was detected. Based on the methylation status of the genomic DNA, selective detection of the parental alleles for H19FRs was examined by using two types of enzymes, the methylation-sensitive restriction enzyme (msRE) HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This parentally imprinted allele (PIA) typing method could be one of the useful techniques for discriminating the parental origin of alleles.

First Report of Phaeoacremonium scolyti Causing Petri Disease of Grapevine in Spain

In May 2007, a survey was conducted to evaluate the phytosanitary status of grapevine propagating materials in a commercial nursery located in Valencia Province (eastern Spain). Fungal isolation was performed on 25 grafted plants (1-year-old grapevines cv. Tempranillo grafted onto 110 R rootstock) because they showed reduced root biomass and black discoloration of the xylem vessels. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were placed onto malt extract agar (MEA) supplemented with streptomycin sulfate (0.5 g L-1). Plates were incubated at 25°C in the dark for 14 to 21 days after which all colonies were transferred to potato dextrose agar (PDA). Togninia minima (Tul. & C. Tul.) Berl. (anamorph Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai) and another Phaeoacremonium sp. were consistently isolated from necrotic tissues. Single conidial isolates of this Phaeoacremonium sp. were grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were grayish brown on PDA and pinkish white on MEA. Conidiophores were mostly short and unbranched, 15 to 30 (mean 20.8) μm long, often consisting of an elongate-ampuliform phialide. Conidia were hyaline, oblong-ellipsoidal occasionally reniform or allantoid, 2.5 to 5.6 (mean 3.8) μm long, and 1 to 2.1 (mean 1.4) μm wide. On the basis of these characteristics, these isolates were identified as Phaeoacremonium scolyti L. Mostert, Summerb. & Crous (2,3). Identity of isolate Psc-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region using Phaeoacremonium-specific primers Pm1-Pm2 and restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the β-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EU260415). The sequence showed high similarity (98%) with the sequence of P. scolyti (GenBank Accession No. AY579292). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using the isolate Psc-1. Ten seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (106 conidia mL-1) harvested from 21-day-old cultures grown on PDA. Ten controls plants were inoculated with sterile distilled water. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months of inoculation, symptoms developed on all of the inoculated plants as crown necrosis, chlorotic leaves, severe defoliation, and wilting. Control plants did not show any symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. scolyti causing Petri disease in Spain.

Analysis of the progression of intraductal proliferative lesions in the breast by PCR-based clonal assay

To analyze the progression in patients with a morphological diagnosis of intraductal proliferative lesions by PCR-based clonal assay. An X-chromosome inactivation assay was applied to explore clonal relationships in human intraductal proliferative lesions of the breast. Four groups samples, including 40 cases of usual ductal hyperplasia (UDH), 40 cases of atypical ductal hyperplasia (ADH), 29 cases of flat epithelia atypia (FEA), and 40 cases of ductal carcinoma in situ (DCIS) were selected for analysis. Thirty specimens of normal breast tissue were used as a control group. Microdissection was performed to collect the tissue samples for extraction of genomic DNA from paraffin-embedded tissues. The DNA was subjected to PCR amplification of the CAG repeats in androgen receptor (AR) gene exon I with and without prior digestion of methylation-sensitive restriction enzyme HhaI. Gel electrophoresis was used to detect the clonal nature of these four groups samples. The clonal analysis confirmed monoclonality in all informative samples of DCIS cells. Normal tissues and the majority (97.1%) of UDH were shown to be polyclonal. Monoclonality was revealed in 20/39 (51.3%) cases of ADH. Among 26 cases of FEA, 20 were shown to be polyclonal, while six displayed monoclonal alterations which accounted for 23.1%. These findings reinforce recent suggestions that clonal analysis with AR gene polymerase chain reaction may be used to define the genetic relationships among the human tumor and the breast intraductal proliferative lesions. Furthermore, our observations demonstrate nearly a half ADH and the smaller part of FEA have clonal alterations, which may be neoplastic lesions. This method would shed light on genetic abnormalities that play a role in early tumorigenesis of the breast, and thus might be an adjunct in predicting the probability of breast tumor occurrence and in guiding the management of these cases.

First Report of Cactodera milleri in Wisconsin.

During the summer of 2005, lemon-shaped cysts and second-stage juveniles of a cyst nematode were recovered from soil at the University of Wisconsin Agricultural Research Station in Hancock, WI. Samples were collected on multiple dates from a plot (61 × 12 m) in continuous potato production for 20 years with significant weed pressure. PCR-restriction fragment length polymorphism profiles of the internal transcribed spacer (ITS) 1 region using restriction enzyme HhaI indicated Cactodera spp. (2). Morphological observations and morphometrics made on cysts, males, J2s, and eggs were consistent with Cactodera milleri Graney and Bird, 1990 (1). Host range studies were conducted in a growth chamber. Soybean, potato, and beet did not support nematode development and reproduction. Common lambsquarters (Chenopodium album), a known host of C. milleri, was an excellent host. No obvious aboveground disease symptoms were evident on lambsquarters in the growth chamber assay. This detection represents the first record of C. milleri in Wisconsin. Unless detailed morphological or molecular measurements are made, C. milleri may be easily confused with the soybean cyst nematode, Heterodera glycines. The presence of lambsquarters in fields planted with glyphosate-resistant soybeans makes the recovery of both nematode species in a single soil sample possible.

Newly developed system based on multiple enzyme restriction fragment length polymorphism – an application to proteolytic bacterial flora analysis

To assist assignment of bacterial phylogeny based on multiple enzyme restriction fragment length polymorphism (MERFLP) analysis, new programs were developed; one program was used to construct reference database of theoretical multiple enzyme restriction fragment (MERF) data, which were edited from DNA sequence of various kinds of bacteria; the other program was used to search the theoretical MERF having the highest similarity to the measured MERF based on pairwise distance according to Nei and Li. Using proteolytic bacteria (96 strains) isolated from various field soils and 25 reference strains, the factors that affected accurate phylogenetic estimation were evaluated. Bacterial strains from the genus Bacillus were successfully separated into species using the MERF with HaeIII and HhaI as the restriction enzymes. Genera Xanthomonas, Pseudomonas, and Alcaligenes were affiliated with the corresponding genus, but precise affiliation under genus level was not always possible. Differentiation among genera in the Enterobacteriaceae may be possible using the other restriction enzymes (HhaI, RsaI and ScrF1). The method was found to be useful for the analysis of bacterial flora because the differentiation, grouping and phylogeny assignment of bacteria, including various genera, were easily accomplished without preliminary information. Most (97.8%) of the proteolytic bacteria isolated from field soils applied with chemical fertilizer or no fertilizer were affiliated with Bacillus spp., whereas the ratios were low in those isolated from field soils applied with liquid livestock feces.

First Report of Purple Coneflower Phyllody Associated with a 16SrI-B Phytoplasma in Maryland.

Purple coneflower (Echinacea purpurea (L.) Moench) is a flowering perennial plant native to North America and is widely grown as an ornamental flower. It is also grown commercially to make herbal teas and extracts purported to help strengthen the immune system. Propagation is by seed or root cuttings. Aster yellows phytoplasmas (strains belonging to group 16SrI) have been reported to be associated with purple coneflower exhibiting virescence and phyllody symptoms in the northern United States and Canada. A subgroup 16SrI-A phytoplasma was identified to be associated with symptomatic purple coneflower in Wisconsin (2). During the summers of 1994 and 2007, purple coneflower plants in Maryland sporadically exhibited symptoms resembling those caused by phytoplasma infection. Symptoms included stunting, virescence, phyllody, and abnormal flower bud proliferation from the cone. Samples from four symptomatic and two asymptomatic purple coneflower plants were collected. Total nucleic acid was extracted from leaf tissue. To assess the etiology of the disease, nested PCR with universal phytoplasma primer pair P1/P7 followed by R16F2n/R16R2 was employed for the detection of phytoplasmas (1). An amplicon of approximately 1.2 kb was amplified from all four symptomatic purple coneflower plants but not from the two asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns of 16S rDNA digested singly with restriction enzymes AluI, KpnI, HpaI, MseI, HhaI, and RsaI indicated that affected purple coneflower plants were infected by a phytoplasma belonging to aster yellows group 16SrI ('Candidatus Phytoplasma asteris'-related strains), subgroup 16SrI-B (1). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU333394 and EU333395) confirmed the results from RFLP analyses. To our knowledge, this is the first report of a 16SrI-B phytoplasma infecting an Echinacea sp. in Maryland.

Molecular Detection and Identification of Group 16SrI and 16SrXII Phytoplasmas Associated with Diseased Potatoes in Russia

Phytoplasmal diseases have long been suspected to occur in several potato-growing regions in Russia on the basis of symptoms and the presence of insect vectors. Symptoms resembling stolbur are most prevalent, but round leaf disease, potato witches'-broom, and potato purple top wilt also occur (1). The phytoplasma etiologies of these diseases have never been verified by molecular means. During the summer of 2006, 33 potato plants exhibiting various symptoms including purple top, round leaves, and stolbur-like symptoms characterized by purple top, stunting, bud proliferation, and formation of aerial tubers were randomly collected from the Volga River Region, Central Region, and Northern Caucasian Region in Russia. DNA extracts were prepared from 1.0 g of petioles and leaf mid veins according to a modified procedure with the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as previously described (2). A nested PCR with primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasmas in infected potato samples (4). Potato plants maintained in the greenhouse were used as healthy controls. A negative control devoid of DNA templates was included in all PCR assays. One microliter of diluted (1:30) PCR product from the first amplification was used as the template in the nested PCR. Eight of 33 potato samples tested positive in the first PCR. Twelve of 33 potato samples tested positive in nested PCR. Nine of the 12 potato samples that tested positive for phytoplasma exhibited stolbur-like symptoms; the other three samples exhibited round leaves, stunting, or proliferation. The remaining symptomatic samples that exhibited nonspecific purple or yellow discoloration of terminal leaves, without other specific stolbur-like symptoms, may be infected by other pathogens. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products (R16F2n/R16R2n amplicons, 1.2 kb) was performed. PCR products (6 μl) were digested singly with the restriction enzymes AluI, HaeIII, HhaI, HpaII, KpnI, MseI, RsaI, and Tsp509I. Comparison of RFLP profiles with published profiles (3) was used for identification of the putative phytoplasmas detected. Among the 12 PCR positive potato samples, 10 showed very similar or identical RFLP profiles to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A and closely related strains, and two showed RFLP profiles similar to those of aster yellows phytoplasma group (16SrI). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU344884-EU344890 and EU333396-EU333400) confirmed the results of the RFLP analyses and also indicated that the two samples showing 16SrI profiles were simultaneously infected with two phytoplasma strains belonging to subgroups 16SrI-A and 16SrI-B. To our knowledge, this is the first confirmation by molecular procedures that stolbur phytoplasma (16SrXII-A) is prevalent in several potato-growing regions and is the first report of 16SrI-A and 16SrI-B phytoplasmas associated with potatoes in Russia.

DNA Hypermethylation Contributes to Incomplete Synthesis of Carbohydrate Determinants in Gastrointestinal Cancer

It has long been known that malignant transformation is associated with abnormal expression of carbohydrate determinants. The aim of this study was to clarify the cause of cancer-associated abnormal glycosylation in gastrointestinal (GI) cancers. We compared the expression levels of "glyco-genes," including glycosyltransferases and glycosidases, in normal GI mucosa and in gastric and colorectal cancer cells. To examine the possibility that DNA hypermethylation contributed to the down-regulation of these genes, we treated GI cancer cells with 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferase. The silencing of some of these glyco-genes, but not up-regulation of certain molecules, was observed. The Sd(a) carbohydrate was abundantly expressed in the normal GI mucosa, but its expression was significantly decreased in cancer tissues. When human colon and gastric cancer cells were treated with 5-aza-dC, cell surface expression of Sd(a) and the transcription of B4GALNT2, which catalyzes the synthesis of the Sd(a), were induced. The promoter region of the human B4GALNT2 gene was heavily hypermethylated in many of the GI cancer cell lines examined as well as in gastric cancer tissues (39 out of 78 cases). In addition, aberrant methylation of the B4GALNT2 gene was strongly correlated with Epstein-Barr virus-associated gastric carcinomas and occurred coincidentally with hypermethylation of the ST3GAL6 gene. Epigenetic changes in a group of glycosyltransferases including B4GALNT2 and ST3GAL6 represent a malignant phenotype of gastric cancer caused by silencing of the activity of these enzymes, which action may eventually induce aberrant glycosylation and expression of cancer-associated carbohydrate antigens.

Genetic Diversity of Heat-Labile Toxin Expressed by Enterotoxigenic Escherichia coli Strains Isolated from Humans

The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

X-chromosomal inactivation analysis of uterine leiomyomas reveals a common clonal origin of different tumor nodules in some multiple leiomyomas

Uterine leiomyomas were shown to be clonal lesions, but the relationship among different tumor nodules in multiple uterine leiomyomas remains unresolved. In this study, X-chromosomal inactivation patterns of these tumor nodules were shown by allelic polymorphism analysis through polymerase-chain reaction at the phosphoglycerate kinase and androgen receptor loci following pretreatment with the methylation-sensitive restriction enzyme HpaII or HhaI. A total number of 113 cases of uterine leiomyomas were examined. Monoclonality was demonstrated in all of the 315 nodules from 76 informative cases. The inter-nodular relationship was evaluated in 55 multiple cases with 294 tumor nodules. Different inactivation patterns were observed in 20 cases, demonstrating a multicentric origin, while an identical inactivated allele was found in all or most of the nodules in the rest of the cases, indicating a common clonal origin. The occurrence of the unicentric cases appeared to be associated with an elevated mitotic activity. Seven nodules from a multinodular case with a morphology indicative of mitotically active leiomyoma were shown to carry the identical inactivated allele, which demonstrates their unicellular origin and malignant nature. In addition, the same androgen receptor gene alteration was identified in two discrete leiomyoma nodules from a uterus. These results approve the monoclonality of uterine leiomyomas and demonstrate the presence of unicentric multiple leiomyomas.

Genetic analysis of the Paramecium aurelia species complex (Protozoa: Ciliophora) by classical and molecular methods

The Paramecium aurelia complex includes 15 species (sibling species) and is characterised by inbreeding (to varying degrees in different species), causing an increase in intra‐specific differentiation. Investigations into inter‐ and intraspecific differentiation of strains originating from remote habitats within species of the complex were carried out by classical inter‐strain crosses and molecular analyses (RAPD‐PCR fingerprints, ARDRA riboprints, RFLP‐PCR analysis). RAPD analysis showed that all species in the complex possessed characteristic band patterns and the majority were also polymorphic intra‐specifically. A correlation exists between the degree of inbreeding characteristic for a species with differentiation of DNAgenotypes revealed by RAPD analysis within species, where inbreeders showed substantial variability of band patterns and moderate inbreeders were highly similar. RFLP analysis (a 480bp fragment of the gene coding the Hsp 70 protein) with the application of restriction enzyme TruII distinguished among species, while digestion with restriction enzyme AluI distinguished groups of species (clusters) and both enzymes revealed intra‐species polymorphism within P. dodecaurelia. ARDRA riboprinting (using a fragment of SSU‐LSU rDNA, about 2400 bp) with restriction enzymes HhaI, AluI, HinfI, TaqI distinguished groups of species with different band patterns. The majority of enzymes also demonstrated intra‐specific differentiation within P. dodecaurelia. TaqI also revealed intraspecific differences in P. biaurelia and P. tetraurelia. All species in the P. aurelia complex showed a high percentage of surviving hybrid clones in F 1 obtained by conjugation and F2 obtained by autogamy in inter‐strain crosses. A low percentage was observed only in F2 inter‐strain hybrids of P. tredecaurelia, however no cytological changes in the nuclear apparatuses were detected and similar band patterns existed in the studied strains. Future studies, including sequencing of rDNA fragments, may disclose deeper relationships of the species.

Identification of Early p53 Mutations in Clam Ileocystoplasties Using Restriction Site Mutation Assay

Because a risk of cancer arising in enterocystoplasties exists, it is necessary to identify which patients are most at risk of tumor formation. The aim of this study was to determine whether rare mutated p53 sequences were more common at the enterovesical anastomosis than in the bladder remnant in patients with a clam ileocystoplasty using the restriction site mutation (RSM) assay. DNA was extracted from endoscopic biopsies obtained from the ileovesical anastomosis and native bladder remnant (control specimens) of 38 patients with a clam ileocystoplasty. The RSM assay was used to study five known hotspots for mutations of the p53 gene using the restriction enzymes Hha I (codon 175), Taq I (codon 213), Hae III (codon 249/250), and Msp I (codons 248 and 282). The mutational events of p53 were confirmed by sequencing the undigested mutated polymerase chain reaction products identified by RSM analysis. We found p53 mutations at the ileovesical anastomosis in 7 of the 38 patients. The mutations were observed at codon 213 (n = 1), codon 248 (n = 3), and codon 250 (n = 3). No p53 mutations were detected in any control specimen. The ileovesical anastomosis is genetically unstable in patients with a clam ileocystoplasty. The p53 mutations identified by the RSM assay at the enterovesical anastomosis could possibly be used as markers of genetic instability to identify patients at risk of developing a tumor. Prospective, randomized longitudinal studies are required to substantiate this hypothesis.

First Report of Phaeoacremonium mortoniae Causing Petri Disease of Grapevine in Spain.

In May 2006, symptoms of grapevine decline were observed on 4-year-old grapevines (cv. Cabernet Sauvignon) grafted onto 110 R rootstock in Daimiel (Ciudad Real Province, central Spain). Affected vines had low vigor, reduced foliage, and chlorotic leaves. Cross or longitudinal sections of the rootstock trunk showed black spots and dark streaking of the xylem vessels. Five symptomatic plants were collected and analyzed for fungal isolation. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g L-1 of streptomycin sulfate. Plates were incubated at 25 to 26°C in the dark for 14 to 21 days and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were yellowish white on PDA and white-to-pale gray on MEA. Conidiophores were short and unbranched, 12.5 to 37.5 (20.5) μm long, and often consisting of a single subcylindrical phialide. Conidia were hyaline, oblong to ellipsoidal or reniform, 2.5 to 7.5 (4.6) μm long, and 1.2 to 1.9 (1.6) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium mortoniae (2,3). Identity of isolate Pmo-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region (Phaeoacremonium-specific primers Pm1-Pm2) with the restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the β-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EF517921). The sequence was identical to the sequence of P. mortoniae (GenBank Accession No. DQ173109). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using two isolates, Pmo-1 and a reference isolate of P. mortoniae (CBS-101585) obtained from the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (106 conidia mL-1) harvested from 21-day-old cultures grown on PDA. Controls were inoculated with sterile distilled water. There were 20 replicates for each isolate with an equal number of uninoculated plants. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months after inoculation, symptoms developed as reduced growth, chlorotic leaves, severe defoliation, and finally wilting. Control plants did not show any of these symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. mortoniae causing young grapevine decline in Spain.