Arbitrarily Primed Polymerase Chain Reaction Research Articles

Isolation and transcript analysis of two-component histidine kinase gene Le.nik1 in Shiitake mushroom, Lentinula edodes

Le.nik1, a two-component histidine kinase gene of Lentinula edodes, the Shiitake mushroom, was identified. The relationship between this two-component signal transduction system and mushroom development was studied. We used a modified RNA arbitrarily-primed PCR (RAP-PCR) method to isolate Le.nik1 as a differentially expressed gene during L. edodes development. We determined the 6.29 kb full-length cDNA sequence of Le.nik1. It had high sequence homology to Neurospora crassa nik1, which encoded a histidine kinase essential for development and osmotic response. In L. edodes, the expression level of Le.nik1 was highest during primordium formation and fruiting body maturation. The transcripts were localized predominantly in the developing hymenophores, or mushroom gills, which may indicate the role of a two-component signal transduction system in cell differentiation during mushroom development. Mannitol stress influenced transcript expression of Le.nik1, suggesting that it may be involved in osmo-sensing and regulation. To our knowledge, this is the first report on the two-component system in mushrooms and the first analysis on the distribution of Le.nik1 transcript in the course of fruiting body formation and in parts of fruiting bodies.

Clinical and epidemiological characterization of infections due to imipenem resistant Acinetobacter baumannii at the university hospital Sahloul, Tunisia

to characterize epidemiological and clinical features related to the multi-drug Acinetobacter baumannii infections in the university hospital Sahloul in Tunisia. retrospective study including twenty-four imipenem resistant Acinetobacter baumannii isolated from twenty patients hospitalized in different wards of the hospital. Study of clinical features related to the infection by multi-drug Acinetobacter baumannii, bacterial identification by classical identification scheme, antibiotic susceptibilities were determined by the disk diffusion method; genotyping was performed by arbitrarily-primed PCR. the most incriminated ward was the intensive care unit with a high prevalence of septicaemia. All studied strains were multi-drug to all beta-lactams tested. Genotyping has shown the clonality of studied strains. Features incriminated in the acquisition of infection were essentially immunodeficiency, invasive manoeuvring and antibiotherapy. multidrug Acinetobacter baumannii is increasingly isolated in our hospital. Rational use of antibiotics and rigorous application of hygienic rules could contribute to limit dissemination of such strains.

Genetic fingerprinting and identification of differentially expressed genes in isolates ofLeishmania donovanifrom Indian patients of post-kala-azar dermal leishmaniasis

Post-kala-azar dermal leishmaniasis (PKDL) is an unusual dermatosis that develops as a sequel in 5-15% of cured cases of kala-azar (KA) after months or years of treatment in India. Molecular differences are reported to exist between the KA and PKDL isolates which may underlie the diversity in clinical manifestations of the disease. Here, arbitrary primed-PCR (AP-PCR) has been used for genetic fingerprinting of parasite isolates from dermal lesions of PKDL patients (n=14) and compared with bone-marrow derived parasites from KA patients (n=3). All isolates showed an identical AP-PCR pattern with 4 arbitrary primers. Further, AP-PCR was exploited to identify the stage regulated genes of the parasite. Six polymorphic fragments were identified in PKDL in comparison with KA isolates, and were subjected to Northern blot analysis. Five polymorphic fragments represented transcribed sequences; 4 out of 5 drew differential expression in pro- and amastigote stages, although the expression was comparable between PKDL and KA isolates. The study led to the identification of genes, which exhibit stage-regulated expression in Leishmania donovani derived from PKDL or KA patients, including a putative phosphodiesterase, DEAD box RNA helicase, iron superoxide dismutase b (fesodb) and a hypothetical protein. Demonstration of transcripts of DEAD box RNA helicase in PKDL and KA diseased tissues implicates its role in disease pathogenesis.

Population genetics of the wood‐decay fungus Phlebia centrifuga P. Karst. in fragmented and continuous habitats

The basidiomycete Phlebia centrifuga is a wood-decay fungus characteristic for unmanaged old-growth forests of spruce, a habitat that has become increasingly fragmented due to forest management. The aim of this study was to investigate the genetic population structures of P. centrifuga in both continuous and fragmented habitats, and estimate the potential impact of fragmentation on the genetic diversity of the fungus. Three hundred fifteen single spore isolates (representing 47 spore families and 33 single isolates) from eight populations across northern Europe (Russia, Finland, and Sweden) were screened with seven microsatellite markers and arbitrary primed polymerase chain reaction with the M13 minisatellite. The two molecular methods generally gave the same pattern for the genetic population structure. There were no significant differences between the observed and the expected heterozygosities, and the inbreeding coefficient (FIS) did not indicate any inbreeding. The fixation index (FST) revealed a general pattern with little to moderate genetic differentiation for the majority of populations, while the southernmost Swedish population Norra Kvill was the only one showing high differentiation from about half of the other populations. Swedish population Fiby with the shortest distance to the continuous habitat was moderately differentiated from most of the others and to the largest extent differed from geographically closest population of Norra Kvill. The results indicate that the fragmentation of old-growth forest in Russia and Finland is more recent than the fragmentation in Sweden, and the genetic population structures of P. centrifuga in northern Europe might be related to differences in forest landscape dynamics between the two areas.

Differential Genetic Expression in Heap Bioleaching of Low-Grade Copper Sulphide Ore at Escondida Mine, Chile

During the last decades, microbial bioleaching of metallic sulphide ores has become a very important process in the mining industry. The study of biodiversity in those systems has revealed the occurrence of several acidophilic organisms, forming a dynamic community including Bacteria and Archaea. Despite the importance of the heap bioleaching process, the metabolic behaviour of a microbial community in bioleaching systems remains unknown. The role played by members of the community inhabiting a bioheap environment has been estimated by the phenotypic characteristics of pure cultures, such as iron oxidation, sulphur oxidation, resistance to high ion concentration and metal tolerance, among others. The global genomic expression of the community inhabiting an industrial bioheap of low-grade copper sulphide ore at Escondida mine in Chile was investigated by random arbitrary primed polymerase chain reaction (RAP-PCR). Three random primers were used and twenty differentially expressed bands were cloned and sequenced. Several sequences were related to 16S rRNA of members of the microbial community; two sequences were related to the α subunit of pyruvate dehydrogenase from Acidithiobacillus ferrooxidans. The expression levels of the pyruvate dehydrogenase gene in samples from an industrial bioleaching operation at different stages were checked by quantitative real-time PCR.

New Insights into a Controversial Topic: The Methylation–Cancer Connection

New Insights into a Controversial Topic: The Methylation–Cancer Connection

Genotypic diversity of Streptococcus mutans in 3- to 4-year-old Chinese nursery children suggests horizontal transmission

Objective The aims of this study were to investigate the colonisation of Streptococcus mutans and to determine the possibility of horizontal transmission in Chinese nursery children. Design Fifty-six children aged between 3 and 4 years old from a nursery were included in this study. Twenty-four children were from the day and night nursery class, the others were from the day nursery class. Dental plaque samples were collected with sterile toothpicks and cultured on S. mutans-selective tryptone-yeast-cystine agar supplemented with 0.2 U/ml bacitracin and 15% sucrose. The typical isolates of S. mutans were identified by classical microbiological methods and genotyped by arbitrarily primed PCR (AP-PCR) and restriction fragment length polymorphism analysis. Results S. mutans was isolated from 41 of the 56 children. The prevalence of S. mutans was higher in the children from day nursery class, compared with the children from day and night nursery class ( P < 0.05). A total of 140 S. mutans isolates from 41 children were analysed by AP-PCR, and 88 different amplitypes were identified. Of 41 children with S. mutans isolates, 82.9% carried two or more genotypes. Two genotypes of S. mutans were repeatedly isolated among 13 children in the day and night nursery class, and one genotype was isolated from two children in the day nursery class. Conclusion The presence of matching genotypes of S. mutans among children attending the same nursery suggests the possibility of horizontal transmission.

The influence of molecular oxygen exposure on the biology ofPrevotella intermedia, with emphasis on its antibiotic susceptibility

This study focuses on investigating the molecular and physiological characteristics of Prevotella intermedia after molecular oxygen exposure (MOE) and the effect on drug susceptibility patterns. Samples of P. intermedia were used as parent strains: ATCC25611 and four clinical isolates. Strains adapted to oxidative stress by MOS were obtained by the enrichment technique. Drug susceptibility was evaluated by minimal inhibitory concentrations (MIC) using agar dilution. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to evaluate the genetic diversity of all strains and physiological analyses were made by sodiumdodecylsulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of crude, cell-free extracts. The genetic profile showed that lineages with altered MIC values were selected after MOE. Overall, we found significant decrease in drug susceptibility for the aero-strains against all tested antimicrobials (amoxicillin, amoxicillin+clavulanic acid, clindamycin, chloramphenicol, ertapenen and metronidazole). We also observed markedly different protein expression patterns between the parent and selected aero-strains. MOE induces changes in the genetic profile and protein expression patterns of P. intermedia that may also be linked to its drug resistance mechanisms. The effects of MOE on anaerobic bacterial physiology and behaviour may influence antimicrobial susceptibility patterns with potential consequences to antimicrobial chemotherapy.

Arbitrarily primed-PCR detection of Streptococcus mutans and Streptococcus sobrinus in dental plaque of children with high dmft and no caries

To isolate and identify Streptococcus mutans (Sm) and Streptococcus sobrinus (Ss) in dental plaque of children with high dmft and no caries by selective medium, biochemical methods and arbitrarily primed-polymerase chain reaction (AP-PCR). A total of 401 3-4-year-old children from seven kindergartens were recruited using cluster sampling and their dental caries status were examined. From 30% of children with the highest dmft score (dmft >/= 5), 20 children were chosen randomly as test group and 20 age and gender-matched caries-free children were selected as control. Plaque samples were collected from buccal surfaces of the molars and plated onto TYCSB plate. Sm and Ss were primarily identified by colony morphology and biochemical characteristics. Then chromosomal DNA of the strains was isolated and Sm or Ss were confirmed by AP-PCR. The proportion positive for Sm and Ss in children with high dmft was 100% and 40% respectively while that in caries-free children was 75% and 5% by AP-PCR analysis. The differences were statistically significant between the two groups. The proportions positive for Sm and Ss detected by AP-PCR method were significantly higher in children with high dmft than in caries-free children and it is a risk factor for high dmft in deciduous teeth harboring Sm and Ss.

Sheep flock infections with Mycoplasma ovipneumoniae involve multiple strains

Infections with Mycoplasma ovipneumoniae have been characterized as occurring with multiple strains within a flock of sheep. In this study clonal isolates of M. ovipneumoniae recovered from 35 lambs from five flocks in central Iowa were analyzed. Using a single primer arbitrarily primed polymerase chain reaction (AP-PCR), and amplified-fragment length polymorphism (AFLP) techniques, a genomic fingerprint was created for each M. ovipneumoniae clonal isolate recovered. The amplified products were separated using gel electrophoresis for the AP-PCR and a sequencer for the AFLP. Band patterns were then analyzed to create dendrograms. In each flock more strains of M. ovipneumoniae were detected when using AP-PCR compared to AFLP. Genomic fingerprints created from the AP-PCR technique yielded fewer bands than those from AFLP. Overall, it was confirmed that multiple strains of M. ovipneumoniae are involved in each flock infection.

Assessment of Genotoxic Activity of Para-nitrophenol in Higher Plant Using Arbitrarily Primed- polymerase Chain Reaction (AP-PCR)

Paranitrophenol is a common toxic environmental pollutant; the aim of this study was to evaluate the potential of the DNA fingerprinting by AP-PCR assay to detect the DNA damage in the common bean (Phaseolus vulgaris) exposed to different concentrations of paranitrophenol (4.0-720 nM). The changes occurring in the fingerprint patterns were likely to be the result of paranitrophenol-induced DNA damage. These changes include variation in band loss and gain. Paranitrophenol was able to induce DNA damage in concentration-related manner with effectiveness at higher concentrations. A total of 488 bands were clearly identified and 39% markers were polymorphic. Genetic distance between control and exposed plant samples served to produce a dendrogram. The dendrogram comprised three main clusters, one of which including control and plant samples exposed to lower concentrations and the other two clusters included plants exposed to higher concentrations. This study clearly demonstrates that AP-PCR is highly useful for assessing DNA damage in plant exposed to chemicals. To the best of our knowledge, this is the first report of the applicability of paranitrophenol for inducing DNA damage in higher plants.

Genotypic diversity of S. mutans in dental biofilm formed in situ under sugar stress exposure

In situ dental biofilm composition under sugar exposure is well known, but sugar effect on the genotypic diversity of S. mutans in dental biofilm has not been explored. This study evaluated S. mutans genotypic diversity in dental biofilm formed in situ under frequent exposure to sucrose and its monosaccharide constituents (glucose and fructose). Saliva of 7 volunteers was collected for isolation of S. mutans and the same volunteers wore intraoral palatal appliances, containing enamel slabs, which were submitted to the following treatments: distilled and deionized water (negative control), 10% glucose + 10% fructose (fermentable carbohydrates) solution or 20% sucrose (fermentable and EPS inductor) solution, 8x/day. After 3, 7 and 14 days, the biofilms were collected and S. mutans colonies were isolated. Arbitrarily primed polymerase chain reaction (AP-PCR) of S. mutans showed that salivary genotypes were also detected in almost all biofilm samples, independently of the treatment, and seemed to reflect those genotypes present at higher proportion in biofilms. In addition to the salivary genotypes, others were found in biofilms but in lower proportions and were distinct among treatment. The data suggest that the in situ model seems to be useful to evaluate genotypic diversity of S. mutans, but, under the tested conditions, it was not possible to clearly show that specific genotypes were selected in the biofilm due to the stress induced by sucrose metabolism or simple fermentation of its monosaccharides.

Transmission of Aggregatibacter actinomycetemcomitans between Brazilian women with severe chronic periodontitis and their children

This study evaluated the transmission of Aggregatibacter actinomycetemcomitans (Aa) in women with severe chronic periodontitis and their children. Thirty women (mean age = 36.1+/-6.0 years) who were mothers of at least one child aged 7 to 16 years were enrolled. In order to investigate mother-child transmission of Aa, the children were also evaluated when their mothers were colonized by the bacterium. Subgingival plaque samples of each woman were collected from 3 sites (mean probing depth of 7.3+/-1.2 mm and mean clinical attachment level of 7.9+/-1.5 mm) and pooled in reduced transport fluid (RTF). These samples were processed, inoculated onto TSBV-agar selective medium and incubated at 37 degrees C in microaerophilic atmosphere for 5 days. Aa was identified on the basis of colony morphology, Gram staining, catalase and oxidase reactions. Aa was found in 8 out of 30 women. Therefore, 8 children from these women (mean age= 12 +/- 3.7 years) were evaluated, but Aa was found only in 2 of them. Aa strains of the two mother-child pairs were evaluated by arbitrarily-primed polymerase chain reaction (AP-PCR), although it was not found similarity between the amplitypes of each pair. No Aa transmission was found between Brazilian women with severe chronic periodontitis and their children.

Methylation of the BIN1 gene promoter CpG island associated with breast and prostate cancer

BackgroundLoss of BIN1 tumor suppressor expression is abundant in human cancer and its frequency exceeds that of genetic alterations, suggesting the role of epigenetic regulators (DNA methylation). BIN1 re-expression in the DU145 prostate cancer cell line after 5-aza-2'-deoxycytidine treatment was recently reported but no methylation of the BIN1 promoter CpG island was found in DU145.MethodsMethylation-sensitive arbitrarily-primed PCR was used to detect genomic loci abnormally methylated in breast cancer. BIN1 CpG island fragment was identified among the differentially methylated loci as a result of direct sequencing of the methylation-sensitive arbitrarily-primed PCR product and subsequent BLAST alliance. BIN1 CpG island cancer related methylation in breast and prostate cancers was confirmed by bisulphite sequencing and its methylation frequency was evaluated by methylation sensitive PCR. Loss of heterozygosity analysis of the BIN1 region was performed with two introgenic and one closely adjacent extragenic microsatellite markers.BIN1 expression was evaluated by real-time RT-PCR.ResultsWe have identified a 3'-part of BIN1 promoter CpG island among the genomic loci abnormally methylated in breast cancer. The fragment proved to be methylated in 18/99 (18%) and 4/46 (9%) breast and prostate tumors, correspondingly, as well as in MCF7 and T47D breast cancer cell lines, but was never methylated in normal tissues and lymphocytes as well as in DU145 and LNCaP prostate cancer cell lines. The 5'-part of the CpG island revealed no methylation in all samples tested. BIN1 expression losses were detected in MCF7 and T47D cells and were characteristic of primary breast tumors (10/13; 77%), while loss of heterozygosity was a rare event in tissue samples (2/22 informative cases; 9%) and was ruled out for MCF7.ConclusionBIN1 promoter CpG island is composed of two parts differing drastically in the methylation patterns in cancer. This appears to be a common feature of cancer related genes and demands further functional significance exploration. Although we have found no evidence of the functional role of such a non-core methylation in BIN1 expression regulation, our data do not altogether rule this possibility out.

Development of SCAR markers for identification of stem rust resistance gene Sr31 in the homozygous or heterozygous condition in bread wheat

AbstractThe stem rust resistance gene Sr31, transferred from rye (Secale cereale) into wheat (Triticum aestivum L.) imparts resistance to all the virulent pathotypes of stem rust (Puccinia graminis f. sp. tritici) found in India. Wheat genotypes including carriers and non‐carriers of the Sr31 gene were analysed using arbitrary primed polymerase chain reaction (AP‐PCR). AP‐PCR markers viz. SS30.2580(H) associated with the Sr31 gene and SS26.11100 associated with the allele for susceptibility were identified. Linkage between the markers and phenotypes was confirmed by analysing an F2 population obtained from a cross between a resistant and a susceptible genotype. The markers were tightly linked to the respective alleles. Both the AP‐PCR markers were converted into sequence characterized amplified region (SCAR) markers, viz. SCSS30.2576 and SCSS26.11100 respectively. The markers were validated in two more segregating populations and 49 wheat genotypes. Using both markers it was possible to distinguish the homozygous from the heterozygous carriers of the Sr31 gene in the F2 generation. The markers developed in this study can be used for pyramiding of the Sr31 gene with other rust resistance genes and in marker‐assisted selection.

The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: A critical review

More than 9000 papers using the random amplified polymorphic DNA (RAPD) or related techniques (e.g. the arbitrarily primed polymerase chain reaction (AP-PCR)) have been published from 1990 to 2005. The RAPD method has been initially used to detect polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. Despite their extensive use, these techniques have also attracted some criticisms, mainly for lack of reproducibility. In the light of their widespread applications, the objectives of this review are to (1) identify the potential factors affecting the optimisation of the RAPD and AP-PCR assays, (2) critically describe and analyse these techniques in genotoxicity and carcinogenesis studies, (3) compare the RAPD assay with other well used methodologies, (4) further elucidate the impact of DNA damage and mutations on the RAPD profiles, and finally (5) provide some recommendations/guidelines to further improve the applications of the assays and to help the identification of the factors responsible for the RAPD changes. It is suggested that after proper optimisation, the RAPD is a reliable, sensitive and reproducible assay, has the potential to detect a wide range of DNA damage (e.g. DNA adducts, DNA breakage) as well as mutations (point mutations and large rearrangements) and therefore can be applied to genotoxicity and carcinogenesis studies. Nevertheless, the interpretation of the changes in RAPD profiles is difficult since many factors can affect the generation of RAPD profiles. It is therefore important that these factors are identified and taken into account while using these assays. On the other hand, further analyses of the relevant bands generated in RAPD profile allow not only to identify some of the molecular events implicated in the genomic instability but also to discover genes playing key roles, particularly in the initiation and development of malignancy. Finally, to elucidate the potential genotoxic effects of environmental contaminants, a powerful strategy could be firstly to use the RAPD assay as a screening method and secondly to apply more specific methods measuring for instance DNA adducts, gene mutations or cytogenetic effects. It is also envisaged that these assays (i.e. RAPD and related techniques), which reflect effects at whole genome level, would continue to complement the use of emerging technologies (e.g. microarrays which aim to quantify expression of individual genes).

Genomic alteration is not associated with fatty and clear cell change in hepatocellular carcinomas and its precursor nodular lesions in cirrhotic liver

The aim of this study was to investigate whether fatty and clear cell areas in large regenerative nodules (LRN), dysplastic nodules (DN), and hepatocellular carcinoma (HCC) show higher degree of genomic mutation compared to non-fatty/clear cell area in the same nodule or non-lesional tissue. We examined 22 nodular lesions (9 HCC, 5 DN and 8 LRN) from seven cirrhotic livers removed at transplantation. Frozen sections were used for manual microdissection of areas with fatty/clear cell change. DNA from microdissected tissue was amplified using arbitrarily primed polymerase chain reaction (AP-PCR), and PCR products were run on polyacrilamide gel generating a "fingerprint" band pattern. Autoradiographs were analysed using Adobe Photoshop version 6.0. Fingerprints from lesional tissue were compared to reference tissue and the total number of bands in excess or defect was calculated and divided by the total number of bands identified, obtaining the genomic damage fraction (GDF). Increasing GDF average values were seen from cirrhotic liver (0.13+/-0.04), to LRN (0.16+/-0.1), DN (0.28+/-0.08) and HCC (0.30+/-0.07). A statistically significant difference in GDF values was documented between cirrhotic liver and DN (p=0.008) and HCC (p=0.005) and between HCC and LRN (p=0.02). No significant difference was documented between DN and HCC, and between LRN and cirrhotic liver. Eleven nodules containing fat/clear cell areas were compared to the other 11 nodules without fat/clear cell areas. The GDF was not different between the two groups: 0.29+/-0.11 versus 0.25+/-0.12; p=0.5. The average value of genomic damage fraction between fat/clear cell areas (0.29+/-0.11) and no fat/clear cell areas (0.25+/-0.1) within the same nodules were not significantly different (p=0.11). Fatty and clear cell change in nodular lesions in cirrhotic liver may be an epigenetic phenotypic modification caused by microenvironmental factors such as ischaemia rather than indicating areas of increased malignant potential per se.

Nasal carriage of methicillin-resistant and methicillin-susceptible Staphylococcus aureus in nursing home residents in Bolu,Turkey

This study aimed (a) to provide information on methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus colonization ratio in residents of nursing homes; (b) to determine the effect of hand-washing education given to nursing home residents and employees on nasal carriage of Staphylococcus aureus (NCSA) and (c) to obtain probable risk factors for MRSA colonization of residents in two nursing homes. Seventy-nine volunteers (24 females and 55 males) from Bolu nursing homes were included in the study. Nasal samples were taken with sterile swabs from the anterior nares. Staphylococcus aureus strains were identified using classical methods and genotyping of methicillin resistant strains was done using Arbitrary Primed PCR (AP-PCR). Antibiotic susceptibilities were determined by disk diffusion methods according to NCCLS standards. After first nasal samples were taken, all employees and residents of nursing homes were educated about the methods of hand hygiene over two days. With hand-washing education, the decrease of NCSA rate (initially 43%; after education, 21%) was significant (p < 0.05) while decrease of MRSA carriage (initially 5%, after education 1%) was not (p > 0.05). MRSA carriage was significantly correlated with presence of skin lesions, prior hospitalization within the last six months, and antibiotics usage within the last six months. AP PCR results suggested that residents' carriage of MRSA was the result of the same source. MSSA and MRSA colonization rates were found to be 38% and 5% in nursing homes, respectively. These ratios can decrease with simple precautions like hand-washing after a short education period

PCR fingerprinting of Blastocystis isolated from symptomatic and asymptomatic human hosts

Genomic DNA from 16 Blastocystis hominis isolates comprising of eight asymptomatic isolates (A1-A8) and eight symptomatic isolates (S1-S8) was amplified by arbitrarily primed polymerase chain reaction (AP-PCR) using 38 arbitrary 10-mer primers. Six primers (A10, B5, C20, D1, F6, and F10) generated reproducible DNA fingerprints. AP-PCR amplification revealed similar DNA fingerprints among all symptomatic isolates (S1-S8) with common bands at 850 bp using primer A10, 920 bp using primer B5, and 1.3 kbp using primer D1. Isolates A1, A3, A4, A5, A6, and A7 showed similar DNA banding patterns and all asymptomatic isolates (A1-A8) shared a major band at 1 kbp using primer B5. Isolates A2 and A8 showed distinct DNA banding patterns that differed from the remainder of the isolates. The results of the phylogenetic analyses showed that all symptomatic isolates (S1-S8) formed a clade with >70% similarity among the isolates and which were clearly separate from asymptomatic isolates A1, A3, A4, A5, A6, and A7. Asymptomatic isolates A2 and A8 formed two distinct and separate clades. AP-PCR revealed higher genetic variability within the asymptomatic isolates than within the symptomatic isolates. The present study suggests that AP-PCR can be a valuable method for differentiating between isolates of B. hominis and our results support the hypothesis that our asymptomatic and symptomatic B. hominis isolates may represent two different strains/species with varying pathogenic potential.

SKCG-1: a new candidate growth regulatory gene at chromosome 11q23.2 in human sporadic Wilms tumours

Using arbitrary primed-PCR (AP-PCR), we have identified a novel genetic alteration located at chromosome 11q23.2 and this genetic alteration was common in 38% of the human Wilms tumour samples analysed. Further characterisation by cloning and sequencing of this genomic region revealed that it represents a part of an uncharacterised gene. We have named this gene as Sporadic Kidney Cancer Gene-1 (SKCG-1). Using fluorescence in situ hybridisation (FISH) approach, we established its localisation on the chromosome 11q23.2. Northern analysis revealed the transcript size of SKCG-1 of 2.09 kb and this was further confirmed by full-length cDNA sequence. Sequence analysis revealed an active translation start site (ATG sequence), a polyadenylation signal sequence (AATAAA), and an open reading frame (ORF) encoding a peptide of 124 amino acids in the cDNA sequence of SKCG-1. Analysis of genomic sequence of SKCG-1 revealed a promoter region containing TATA box located at −13 bp upstream of transcription start site. The AP-PCR, SCAR, and Southern blot analyses indicated genomic loss of SKCG-1 in Wilms tumours. The transcript of SKCG-1 was abundantly present in brain, kidney, liver, testis, salivary gland, foetal brain, foetal liver, whereas relatively lower expression in heart, stomach, prostate and no expression in spleen, colon, lung, small intestine, muscle, adrenal gland, uterus, skin, PBL, and bone marrow was detected. The expression of this gene transcript was either very less or undetectable in Wilms and breast tumours compared to their matched uninvolved tissues. Inhibition of SKCG-1 by siRNA resulted in increased cell proliferation of kidney epithelial cells. Based on the presence of two transmembrane regions in its peptide, SKCG-1 has been predicted as a transmembrane protein. Thus, the findings of this study revealed (i) SKCG-1, a new gene located at 11q23.2 and harbouring genetic alteration in Wilms tumours, (ii) the presence of SKCG-1 gene transcripts in various human normal tissues and its lower expression or absence in Wilms and breast tumours indicate that it may be associated with tumour growth suppressor activity, (iii) the presence of an open reading frame in the cDNA sequence of SKCG-1 indicates that it has potential to encode a protein, (iv) increased cell growth by silencing this gene in HEK293 cells further supports a potential role of this gene in growth of kidney epithelial cells. Our findings suggest that SKCG-1 may have a tumour suppressor role, and implicate genetic alteration in this gene as a potential oncogenic pathway and therapeutic target in kidney and breast cancer.