Genomic DNA Restriction Research Articles

Light Dependent Changes in Adenylate Methylation of the Promoter of the Mitochondrial Citrate Synthase Gene in Maize (Zea mays L.) Leaves.

Limited methyl-specific restriction of genomic DNA by endonuclease MAL1 revealed the changes in its methyl status caused by adenine modification in maize (Zea mays L.) leaves under different light conditions (dark, light, irradiation by red and far-red light). Incubation in the light and irradiation by red light exhibited an activating effect on DNA adenine methylase activity, which was reflected in an increase in the number of methylated adenines in GATC sites. Far-red light and darkness exhibited an opposite effect. The use of nitrite conversion of DNA followed by methyladenine-dependent restriction by MboI nuclease revealed a phytochrome B-dependent mechanism of regulation of the methyl status of adenine in the GATC sites in the promoter of the gene encoding the mitochondrial isoform of citrate synthase. Irradiation of plants with red light caused changes in the adenine methyl status of the analyzed amplicon, as evidenced by the presence of restriction products of 290, 254, and 121 nucleotides. Adenine methylation occurred at all three GATC sites in the analyzed DNA sequence. It is concluded that adenylate methylation is controlled by phytochrome B via the transcription factor PIF4 and represents an important mechanism for the tricarboxylic acid cycle regulation by light.

Survey of the association between polymorphisms of CTLA-4 exon 1 49 A/G genes with rheumatoid arthritis in Iran

ABSTRACT Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which suppresses T cell proliferation, is a promising candidate for the susceptibility genes to rheumatic arthritis diseases (RA). This study aims to examine the association between the polymorphisms of the CTLA-4 exon 1(+ 49) genes with RA in the Qazvin city of Iran population. The polymerase chain reaction of genomic DNA-restriction fragment length polymorphism (PCR-RFLP) was applied to genotype the CTLA-4 exon 1(+ 49) polymorphisms in 105 RA patients and 90 control subjects. Laboratory diagnostic tests were also measured for RA and control groups. Our results did not demonstrate a significant difference in allele and genotype frequencies of the CTLA-4 exon 1(+ 49) between RA patients and the control group (p < .0001). There was no significant difference in age at onset, CRP, RF value in patients with RA according to the CTLA-4 polymorphisms; just anti-CCP showed a significant difference. Our data declared that polymorphisms of CTLA-4 exon 1(+ 49) genes are not correlated with RA susceptibility and its clinical and paraclinical manifestations.

Determination of virulence and genetic variability of three Spodoptera littoralis nucleopolyhedrovirus isolates from Egypt

BackgroundThe genetic diversity is naturally occurring in baculovirus population, which may affect the potency of the bio-agent. The Spodoptera littoralis nucleopolyhedrovirus is a promising bio-control agent that can be used to control the cotton leafworm, S. littoralis in Egypt. This study described the virulence and genetic variability of 3 S. littoralis nucleopolyhedrovirus isolates named Cab2, P2 and P3 previously recovered from diseased S. littoralis larvae of cabbage, pepper and eggplant cultivation fields.ResultsThe viral genomic DNA restriction analysis using PstI and ScaI endonucleases showed identical Pst1 restriction pattern of 10 fragments in both Cab2 and P3 isolates, while there are 7 extra fragments present in P2 isolate of about 25, 19.8, 15, 14, 11, 9 and 6.7 (kbp). On the other hand, the restriction pattern using ScaI revealed 10 common fragments in all three isolates, in addition to an extra marker fragment of about 25 kb in P3 isolate absent in both P2 and Cab3 isolates. The phylogenetic analysis of the partial nucleotide sequence of the polyhedrin gene confirmed an extensive degree of co-linearity among the genomes of the 3 tested isolates, with pairwise sequence identities of 97.04% with S. littoralis nucleopolyhedrovirus isolate A26-5 (Acc. No. AY706717). The median lethal concentration (LC50) value revealed that Cab2 had the best virulence toward the first instar of S. littoralis larvae with LC50 value of 1.59 × 104 Obs/ml compared to the virulence recorded by P3 and P2 which were 1.23 × 106 and 2.7 × 104 OBs/ml, respectively. The median survival time (LT50) value of Cab2 isolate was the highest speed of treated larval mortality with 120 hpi compared to P2 and P3, which recorded 132 hpi for both isolates.ConclusionsAlthough all three S. littoralis nucleopolyhedrovirus isolates showed considered genotypic variation and insecticidal activity against S. littoralis larvae, the variant Cab2 showed the highest virulence and speed mortality against the first instar larvae of S. littoralis. The new variant proved to possess promising insecticidal properties for the development of virus-based biopesticide for the control of S. littoralis population in Egypt.

Canine Model For The Study of the Epigenetic Factors Involved in Mammary Tumor Development

Cancer is a multifactorial disease involving reprogramming of the gene function in normal cells through both genetic and epigenetic factors. The latter ones are essentially represented by chromatin remodeling factors as transcriptional control of gene expression at the nucleus level. Certain sets of tumor suppression genes controlling vital cell functions such as cell cycle, DNA repair, cell adhesion, and apoptosis are considered candidate genes for defining specific types of cancer. Their silencing may be achieved through somatic mutations or by epigenetic factors as DNA methylation and histone covalent modification. Two major DNA methylation patterns have been described in mammary gland tumors: genome hypomethylation and concomitant "in situ" hypermethylation in critical genes. Human mammary gland cancer has been studied through animal models, canine one offering opportunity to investigate the molecular aspects that better define the borderline between the primary inflammatory and tumor progression processes. In contrast, both humans and dogs share the same challenging environment. This work presents the preliminary results in a study of the transcriptional epigenetic markers identified in canine mammary glands during tumor development. Bisulfite mutagenesis based methylation-specific (MS)- PCR has been performed for the estimation of BRCA1, and BRCA2 gene promoter hyper-methylation profiles, concomitant to methylation sensible restriction of genomic DNA in order to estimate the global hypo-methylation status have been performed on 9 DNA samples obtained from clinically selected canine tumor tissues. Histone H3K9 trimethylated was stained by immunohistochemical methods (IHC) in normal and tumor tissues. The results are discussed through their crosstalk with DNA methylation dynamics in order to highlight the cancer initiation and progression processes in the context of heritability and the causes of sporadic cases.

PFGE: a tool for examination of heterogeneity between the bacterial spot-causing xanthomonads of tomato plants in Bulgaria.

Pulsed-field gel electrophoresis (PFGE) is a highly discriminative molecular typing method that is used for epidemiological studies and investigation of outbreaks caused by different pathogens, including phytopathogenic Xanthomonas species. Bacterial spot (BS) is the most common and one of the most destructive diseases of tomato and pepper plants in Bulgaria. Several Xanthomonas species are known to cause BS, but the global distribution and genetic diversity of these species are not well understood. A collection of 100 BS-causing strains, isolated during the period of 1985-2012 from different tomato cultivars and weeds associated with tomato production areas from 11 geographic regions in Bulgaria, were screened for genetic diversity by genomic DNA restriction with rare-cutting endonucleases (XbaI and SpeI) subsequently resolved by PFGE. Two haplotypes for Xanthomonas vesicatoria and one haplotype for Xanthomonas gardneri strains were found.

Detection of Cytochrome P450 Polymorphisms in Breast Cancer Patients May Impact on Tamoxifen Therapy

Background:Breast cancer is the most common cancer among women worldwide. Tamoxifen (TAM), a selective estrogen receptor modulator, is widely used in its treatment. TAM is metabolized by cytochrome P450 (CYP450) enzymes, including CYP2D6, CYP3A5 and CYP2C19, whose genetic variations may have clinicopathological importance. However, reports on the association of various P450 polymorphisms with certain cancers are contradictory.Methods:We here investigated whether the prevalence of the four most common polymorphism in the CYP2D6*4 (G1934A), CYP2D6*10 (C188T), CYP3A5*3 and CYP2C19*2 alleles has any link with breast cancer using genomic DNA and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis.Results:Prevalences of CYP2D6*4, CYP2D6*10 and CYP2C19*2 genotypes were differed significantly (P = 0.01 and P = 0.004) between breast cancer patients and controls. The CYP3A5*3 genotype did not demonstrate statistically significant variation.Conclusion:Polymorphisms in CYP2 appear to be associated with breast cancer risk. Our data taken together with other reports indicates that drug resistance gene polymorphisms might be indicators of response to tamoxifen therapy in breast cancer cases.

Multidrug resistant Pseudomonas aeruginosa survey in a stream receiving effluents from ineffective wastewater hospital plants.

BackgroundMulti-drug resistant forms of Pseudomonas aeruginosa (MDRPA) are a major source of nosocomial infections and when discharged into streams and rivers from hospital wastewater treatment plants (HWWTP) they are known to be able to persist for extended periods. In the city of Manaus (Western Brazilian Amazon), the effluent of three HWWTPs feed into the urban Mindu stream which crosses the city from its rainforest source before draining into the Rio Negro. The stream is routinely used by Manaus residents for bathing and cleaning (of clothes as well as domestic utensils) and, during periods of flooding, can contaminate wells used for drinking water.Results16S rRNA metagenomic sequence analysis of 293 cloned PCR fragments, detected an abundance of Pseudomonas aeruginosa (P. aeruginosa) at the stream’s Rio Negro drainage site, but failed to detect it at the stream’s source. An array of antimicrobial resistance profiles and resistance to all 14 tested antimicrobials was detected among P. aeruginosa cultures prepared from wastewater samples taken from water entering and being discharged from a Manaus HWWTP. Just one P. aeruginosa antimicrobial resistance profile, however, was detected from cultures made from Mindu stream isolates. Comparisons made between P. aeruginosa isolates’ genomic DNA restriction enzyme digest fingerprints, failed to determine if any of the P. aeruginosa found in the Mindu stream were of HWWTP origin, but suggested that Mindu stream P. aeruginosa are from diverse origins. Culturing experiments also showed that P. aeruginosa biofilm formation and the extent of biofilm formation produced were both significantly higher in multi drug resistant forms of P. aeruginosa.ConclusionsOur results show that a diverse range of MDRPA are being discharged in an urban stream from a HWWTP in Manaus and that P. aeruginosa strains with ampicillin and amikacin can persist well within it.

Metabolite profiling reveals abiotic stress tolerance in Tn5 mutant of Pseudomonas putida.

Pseudomonas is an efficient plant growth–promoting rhizobacteria (PGPR); however, intolerance to drought and high temperature limit its application in agriculture as a bioinoculant. Transposon 5 (Tn5) mutagenesis was used to generate a stress tolerant mutant from a PGPR Pseudomonas putida NBRI1108 isolated from chickpea rhizosphere. A mutant NBRI1108T, selected after screening of nearly 10,000 transconjugants, exhibited significant tolerance towards high temperature and drought. Southern hybridization analysis of EcoRI and XhoI restricted genomic DNA of NBRI1108T confirmed that it had a single Tn5 insertion. The metabolic changes in the polar and non-polar extracts of NBRI1108 and NBRI1108T were examined using 1H, 31P nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Thirty six chemically diverse metabolites consisting of amino acids, fatty acids and phospholipids were identified and quantified. Insertion of Tn5 influenced amino acid and phospholipid metabolism and resulted in significantly higher concentration of aspartic acid, glutamic acid, glycinebetaine, glycerophosphatidylcholine (GPC) and putrescine in NBRI1108T as compared to that in NBRI1108. The concentration of glutamic acid, glycinebetaine and GPC increased by 34%, 95% and 100%, respectively in the NBRI1108T as compared to that in NBRI1108. High concentration of glycerophosphatidylethanolamine (GPE) and undetected GPC in NBRI1108 indicates that biosynthesis of GPE may have taken place via the methylation pathway of phospholipid biosynthesis. However, high GPC and low GPE concentration in NBRI1108T suggest that methylation pathway and phosphatidylcholine synthase (PCS) pathway of phospholipid biosynthesis are being followed in the NBRI1108T. Application of multivariate principal component analysis (PCA) on the quantified metabolites revealed clear variations in NBRI1108 and NBRI1108T in polar and non-polar metabolites. Identification of abiotic stress tolerant metabolites from the NBRI1108T suggest that Tn5 mutagenesis enhanced tolerance towards high temperature and drought. Tolerance to drought was further confirmed in greenhouse experiments with maize as host plant, where NBRI1108T showed relatively high biomass under drought conditions.

Characterization of Two Temperate Lactobacillus paracasei Bacteriophages: Morphology, Kinetics and Adsorption

Background/Aims: Adsorption and kinetic parameters, latent period, burst size and burst time, are characteristics of phage/host systems and can be affected by several environmental factors. As only few studies have focused on temperate dairy phages, we characterized these parameters on temperate Lactobacillus paracasei phages Φ iLp84 and Φ iLp1308, infective for probiotic strains. Methods: Phages were characterized by transmission electron microscopy and genomic DNA restriction. Adsorption under different environmental conditions, phage kinetics and efficiency of plating (EOP) were determined using the double-layer titration method. Results: Phages Φ iLp84 and Φ iLp1308 belong to the Siphoviridae family and have genome sizes of 38 and 34 kbp, respectively. Adsorption was affected by calcium concentration, pH, temperature and host viability, and reached a limit at very high multiplicity of infection. Latency, burst time and burst size were of 85 min, 131 min and 46 for Φ iLp84, and 51 min, 92 min and 28 for Φ iLp1308, respectively, at 37°C. A clear influence of temperature on phage kinetics was observed. Regarding EOP, Φ iLp84 produced plaques on only 1 out of 8 strains tested. Conclusion: Noticeable differences in adsorption, kinetics and EOP were found for two morphologically identical temperate L. paracasei phages of similar origin.

Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL), a new method to detect translocations in leukemia.

Here we present a novel method “Genomic inverse PCR for exploration of ligated breakpoints” (GIPFEL) that allows the sensitive detection of recurrent chromosomal translocations. This technique utilizes limited amounts of DNA as starting material and relies on PCR based quantification of unique DNA sequences that are created by circular ligation of restricted genomic DNA from translocation bearing cells. Because the complete potential breakpoint region is interrogated, a prior knowledge of the individual, specific interchromosomal fusion site is not required. We validated GIPFEL for the five most common gene fusions associated with childhood leukemia (MLL-AF4, MLL-AF9, MLL-ENL, ETV6-RUNX1, and TCF3-PBX1). A workflow of restriction digest, purification, ligation, removal of linear fragments and precipitation enriching for circular DNA was developed. GIPFEL allowed detection of translocation specific signature sequences down to a 10−4 dilution which is close to the theoretical limit. In a blinded proof-of-principle study utilizing DNA from cell lines and 144 children with B-precursor-ALL associated translocations this method was 100% specific with no false positive results. Sensitivity was 83%, 65%, and 24% for t(4;11), t(9;11) and t(11;19) respectively. Translocation t(12;21) was correctly detected in 64% and t(1;19) in 39% of the cases. In contrast to other methods, the characteristics of GIPFEL make it particularly attractive for prospective studies.

The clinical management of cesarean section-acquired Mycobacterium abscessus surgical site infections

Rapidly growing mycobacteria (RGM) can cause a broad spectrum of both community and healthcare-associated infections in humans. The aim of this study was to report the clinical management and outcomes of successive patients following cesarean delivery with healthcare-associated surgical site infections (SSIs) caused by RGM. Patients who were admitted to Chung Shan Medical University Hospital, Taichung, Taiwan, between September 2006 and July 2008, and who developed SSIs following cesarean delivery at an obstetrics hospital and were then referred to our hospital, were enrolled. Demographic characteristics of the patients and clinical isolates were obtained retrospectively and an environmental investigation was performed. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp65gene and pulsed-field gel electrophoresis (PFGE) of large genomic DNA restriction fragments were applied to differentiate Mycobacterium species. Seventeen patients were diagnosed with RGM infections by microbiology and/or histopathology. Mycobacterial isolates by PCR-RFLP analysis from 15 patients revealed Mycobacterium abscessus (M. abscessus) and M. lentiflavum. Most of the patients received surgical debridement and combination antimicrobial therapy and were eventually cured. Our study demonstrates the potential that RGM infections have in causing healthcare-associated SSIs. Surgery plus prolonged combination antimicrobial therapy seemed to be an effective option for the management of M. abscessus infections.

Using PCR‐RFLP technology to teach single nucleotide polymorphism for undergraduates

Recent studies indicated that the aberrant gene expression of peroxiredoxin-6 (prdx6) was found in various kinds of cancers. Because of its biochemical function and gene expression pattern in cancer cells, the association between genetic polymorphism of Prdx6 and cancer onset is interesting. In this report, we have developed and implemented a serial experiment in molecular biology laboratory course to teach single nucleotide polymorphism (SNP) to undergraduate students majoring in molecular biology or genetics. The flanking sequence of rs4382766 was located in Prdx6 gene, which contained a restriction site of SspI, and was used as a target in this lab course. The students could mimic real research by integrating different techniques, such as database retrieving, genomic DNA isolation, PCR, and restriction enzyme assay. This serial experiment of PCR-RFLP helps students set up intact idea of molecular biology and understand the relation among individual experiments. Students were found to be more enthusiastic during the laboratory classes than those in the former curriculum.

Presence of β-lactamases in extended-spectrum-cephalosporin-resistant Salmonella enterica of 30 different serovars in Germany 2005–11

Between 20 000 and 35 000 cases of salmonellosis are detected annually in Germany, but only a few Salmonella are resistant to third-generation cephalosporins. The German National Reference Centre for Salmonella and other Enterics obtained 150 Salmonella enterica isolates from human infections between 2005 and 2011. In the present study we identified the β-lactamase genes causing resistance to third-generation cephalosporins in these isolates. For all isolates serotyping and antimicrobial susceptibility testing were performed. The presence of β-lactamase genes was detected by PCR amplification and sequencing. Isolates with identical serovar and β-lactamase genes were typed by XbaI macrorestriction followed by PFGE. Broth mate conjugation assays and plasmid analysis using S1 nuclease restriction of genomic DNA and subsequent PFGE as well as PCR-based replicon typing were performed for selected isolates. The 150 isolates were assigned to 30 different serovars, with S. enterica serovar Typhimurium (n = 73; 48.7%) as the most prevalent. Two different AmpC β-lactamase genes (blaCMY-2, n = 8; blaACC-1, n = 6) and various extended-spectrum β-lactamase (ESBL) genes were identified. The majority harboured the blaCTX-M-1 gene (n = 91; 60.7%) followed by blaCTX-M-14 (n = 12; 8.0%) and blaSHV-12 (n = 11; 7.3%). Typing of strains and subsequent comparison with selected Salmonella isolates from livestock revealed the presence of several clones in both humans and livestock. The wide spread of ESBL and AmpC genes in Salmonella of various serovars is most probably due to transfer of conjugative plasmids. Furthermore, our data indicate the clonal spread of distinct cephalosporin-resistant Salmonella strains from livestock to humans.

Isolation and characterization of soilborne virulent bacteriophages infecting the pathogen Rhodococcus equi

To isolate and characterize a diversity of bacteriophages (phages) that infect the soilborne pathogen Rhodococcus equi. Twenty-seven phages were isolated from soil samples from geographically distinct locations using a range of R.equi bacterial strains, including clinical isolates. On the basis of host range, genomic DNA restriction profiles and virion protein profiles, the diversity of these phages was extensive, with phages being divided into 16 groupings. Based on a range of criteria, these phages could be divided into 16 distinct groupings. The majority of the phages recovered from soil were Siphoviridae, adding to the limited number of Siphoviridae described to date for R.equi. One grouping consisted of phages belonging to the Myoviridae. This represents the first study looking at the diversity of phages infecting the pathogen R.equi, including the first Myoviridae to be isolated and characterized for the genus Rhodococcus and for the nonmycobacterial actinomycetes. Given their diverse host range, including clinical isolates, this collection of phages offers the potential for the development of phage cocktails for use as a therapeutic or alternatively in the biocontrol of this pathogen in reservoirs of infection relating to animal husbandry.

Relationship of ERCC1 genotype variant with mRNA expression and ERCC1 protein levels in advanced urothelial carcinoma (UC).

260 Background: DNA repair factors may be predictive for response to chemotherapies that produce DNA damage. While low ERCC1 protein and mRNA levels have been reported as associated with improved outcomes in metastatic UC patients treated with platinum-based chemotherapy, the relationship between genotype, mRNA expression, and protein level is unknown. The ERCC1 germline 19007C&gt;T single-nucleotide polymorphism (SNP) is functionally associated with reduced translation of ERCC1 mRNA. We investigated the relationship between ERCC1 germline SNP, ERCC1 tumor mRNA and protein expression, in a cohort of patients with advanced UC who received first-line, platinum-based chemotherapy. Methods: A cohort of clinically annotated, uniformly-treated advanced UC patients with FFPE primary tumor tissue available was identified through the Hellenic cooperative Oncology Group (HECOG) (N=93). Genomic DNA extraction, nested PCR, and restriction fragment length polymorphism techniques for the 19007C&gt;T SNP were performed to identify C/C, C/T and T/T genotypes. ERCC1 mRNA expression was interrogated using Nanostring nCounter profiling. IHC analysis was performed on tissue arrays using an ERCC1 antibody. Percent of positive nuclear staining was categorized as quartiles using previously identified cut-points. Results: ERCC1 C/T genotype was identified in 30/61 samples (49%) and T/T in 14/61 samples (23%). In 54 patients with both SNP and mRNA data available, T/T genotype was associated with the highest level of mRNA expression, followed by the C/T genotype (p=0.04). Neither ERCC1 genotype (N=44) nor ERCC1 mRNA expression (N=54) was associated with ERCC1 protein expression as measured by IHC (p=0.52 and p=0.13, respectively). Conclusions: ERCC1 19007C&gt;T is associated with increased ERCC1 mRNA expression. However, neither genotype nor mRNA are surrogates for ERCC1 protein detected by IHC in advanced UC tumors. This suggests that while genotype influences mRNA expression of ERCC1, the use of the nucleotide excision repair pathway as a predictive biomarker of platinum-sensitivity may be more complex than previously appreciated and require the integrative use of proteomics, genomics and epigenomics.

Screening and characterization of sex-specific DNA fragments in the freshwater fish matrinchã, Brycon amazonicus (Teleostei: Characiformes: Characidae)

The matrinchã Brycon amazonicus, a commercially important freshwater fish resource, has no heteromorphic sex chromosomes so far described. In the present study, we performed a screening of sex-associated DNA markers in this species, through the use of a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis. DNA digestions evidenced no differences between sexes. Sixty-six random primers were used in pooled and individual DNA samples of males and females, and the analysis of the RAPD fingerprints revealed one female sex-associated band. Cloning and sequencing of this band led to the identification of two distinct DNA segments. While one of the isolated fragments showed a significant identity with a described protein gene (phosphatidylinositol glycan anchor biosynthesis, class W), the other fragment, composed of 535 bp, corresponds to a novel DNA marker. Further experiments were performed with this second DNA fragment in order to verify its sex-specificity. Data on dot blot hybridization, using total DNA of both sexes, confirmed its female-specificity in B. amazonicus. A primer set was designed based on its sequence data and used in PCR with DNA samples of this species, leading to diagnose the animals' sexes with a 100 % overall accuracy through a sequence characterized amplified region approach. No amplification results were found for two other species of the genus--B. orbignyanus and B. lundii. The obtained data can lead to the hypothesis that B. amazonicus may present heteromorphic sex chromosomes that should be in an early phase of differentiation.

Association of the Functional Polymorphism C677T in the Methylenetetrahydrofolate Reductase Gene with Colorectal, Thyroid, Breast, Ovarian, and Cervical Cancers

Background: Polymorphisms of the gene encoding methylenetetrahydrofolate reductase (MTHFR) have been studied widely in various cancers, excluding thyroid cancer. However, reports on the association of various polymorphisms with certain cancers are contradictory. Materials and Methods: We have investigated whether the prevalence of the most common polymorphism (C677T) in the MTHFR gene has any link with various cancers, using genomic DNA and polymerase chain reaction restriction fragment length polymorphism (PCRRFLP) analysis. Results: The frequency of the heterozygous variant (677CT) but not that of 677TT was found to be significantly higher in colorectal cancer cases than in controls (p < 0.039; odds ratio 2.35 and 95% confidence interval 1.02–5.415). The frequencies for 677CT were 11.0 and 5.0% in colorectal cancer samples and controls, respectively. In ovarian cancer, the frequency of the 677TT variant was 6.0% which differed significantly (p < 0.026) from the control value of 1.0%. However, the frequencies of the variants in cervical, thyroid, and breast cancer cases did not differ from controls. Conclusions: Our data taken together with other reports indicates that the polymorphism C677T of MTHFR is a risk factor for developing colorectal cancer but not cervical, thyroid, and breast cancers. The present study also reconfirms that frequencies of the variants are not gender-specific.

Nosocomial outbreak of Blastoschizomyces capitatus associated with contaminated milk in a haematological unit

In July 2002, Blastoschizomyces capitatus was isolated from four neutropenic patients in a haematology unit. Two patients died due to disseminated infection while the other two had oropharyngeal colonisation. Nosocomial acquisition of the fungus was suspected and epidemiological and environmental studies were undertaken. To determine the potential source for the acquisition of the fungus, epidemiological relationships between the patients were investigated. We performed surveillance cultures on all patients and took environmental cultures of air, inanimate surfaces, food samples, blood products and chemotherapy drugs. No direct contact transmission between patients was found and B. capitatus was isolated only in vacuum flasks used for breakfast milk distribution. All isolates were compared by four independent molecular typing methods: pulsed-field gel electrophoresis, genomic DNA restriction endonuclease analysis, randomly amplified polymorphic DNA, and polymerase chain reaction fingerprinting using a single primer specific for one minisatellite or two microsatellite DNAs. Milk vacuum flasks and clinical strains were genetically indistinguishable by all typing techniques. Milk vacuum flasks were withdrawn from all hospital units and no further B. capitatus infection was detected. Our findings suggest that clonal dissemination of a single strain of B. capitatus from vacuum flasks used for milk distribution was responsible for this nosocomial outbreak in the haematological unit.

Identification of two new repetitive elements and chromosomal mapping of repetitive DNA sequences in the fish Gymnothorax unicolor (Anguilliformes: Muraenidae)

Muraenidae is a species-rich family, with relationships among genera and species and taxonomy that have not been completely clarified. Few cytogenetic studies have been conducted on this family, and all of them showed the same diploid chromosome number (2n=42) but with conspicuous karyotypic variation among species. The Mediterranean moray eel Gymnothorax unicolor was previously cytogenetically studied using classical techniques that allowed the characterization of its karyotype structure and the constitutive heterochromatin and argyrophilic nucleolar organizer regions (Ag-NORs) distribution pattern. In the present study, we describe two new repetitive elements (called GuMboI and GuDdeI) obtained from restricted genomic DNA of G. unicolor that were characterized by Southern blot and physically localized by in situ hybridization on metaphase chromosomes. As they are highly repetitive DNA sequences, they map in heterochromatic regions. However, while GuDdeI was localized in the centromeric regions, the GuMboI fraction was distributed on some centromeres and was co-localized with the nucleolus organizer region (NOR). Comparative analysis with other Mediterranean species such as Muraena helena pointed out that these DNA fractions are species-specific and could potentially be used for species discrimination. As a new contribution to the karyotype of this species, we found that the major ribosomal genes are localized on acrocentric chromosome 9 and that the telomeres of each chromosome are composed of a tandem repeat derived from a poly-TTAGGG DNA sequence, as it occurs in most vertebrate species. The results obtained add new information useful in comparative genomics at the chromosomal level and contribute to the cytogenetic knowledge regarding this fish family, which has not been extensively studied.

Satellite DNA and chromosomes in Neotropical fishes: methods, applications and perspectives

Constitutive heterochromatin represents a substantial portion of the eukaryote genome, and it is mainly composed of tandemly repeated DNA sequences, such as satellite DNAs, which are also enriched by other dispersed repeated elements, including transposons. Studies on the organization, structure, composition and in situ localization of satellite DNAs have led to consistent advances in the understanding of the genome evolution of species, with a particular focus on heterochromatic domains, the diversification of heteromorphic sex chromosomes and the origin and maintenance of B chromosomes. Satellite DNAs can be chromosome specific or species specific, or they can characterize different species from a genus, family or even representatives of a given order. In some cases, the presence of these repeated elements in members of a single clade has enabled inferences of a phylogenetic nature. Genomic DNA restriction, using specific enzymes, is the most frequently used method for isolating satellite DNAs. Recent methods such as C(0)t-1 DNA and chromosome microdissection, however, have proven to be efficient alternatives for the study of this class of DNA. Neotropical ichthyofauna is extremely rich and diverse enabling multiple approaches with regard to the differentiation and evolution of the genome. Genome components of some species and genera have been isolated, mapped and correlated with possible functions and structures of the chromosomes. The 5SHindIII-DNA satellite DNA, which is specific to Hoplias malabaricus of the Erythrinidae family, has an exclusively centromeric location. The As51 satellite DNA, which is closely correlated with the genome diversification of some species from the genus Astyanax, has also been used to infer relationships between species. In the Prochilodontidae family, two repetitive DNA sequences were mapped on the chromosomes, and the SATH 1 satellite DNA is associated with the origin of heterochromatic B chromosomes in Prochilodus lineatus. Among species of the genus Characidium and the Parodontidae family, amplifications of satellite DNAs have demonstrated that these sequences are related to the differentiation of heteromorphic sex chromosomes. The possible elimination of satellite DNA units could explain the genome compaction that occurs among some species of Neotropical Tetraodontiformes. These topics are discussed in the present review, showing the importance of satellite DNA analysis in the differentiation and karyotype evolution of Actinopterygii.