Isolation Of DNA Sequence Research Articles

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

AbstractSuspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.

Cluster and sporadic cases of herbaspirillum species infections in patients with cancer.

Herbaspirillum species are gram-negative Betaproteobacteria that inhabit the rhizosphere. We investigated a potential cluster of hospital-based Herbaspirillum species infections. Cases were defined as Herbaspirillum species isolated from a patient in our comprehensive cancer center between 1 January 2006 and 15 October 2013. Case finding was performed by reviewing isolates initially identified as Burkholderia cepacia susceptible to all antibiotics tested, and 16S ribosomal DNA sequencing of available isolates to confirm their identity. Pulsed-field gel electrophoresis (PFGE) was performed to test genetic relatedness. Facility observations, infection prevention assessments, and environmental sampling were performed to investigate potential sources of Herbaspirillum species. Eight cases of Herbaspirillum species were identified. Isolates from the first 5 clustered cases were initially misidentified as B. cepacia, and available isolates from 4 of these cases were indistinguishable. The 3 subsequent cases were identified by prospective surveillance and had different PFGE patterns. All but 1 case-patient had bloodstream infections, and 6 presented with sepsis. Underlying diagnoses included solid tumors (3), leukemia (3), lymphoma (1), and aplastic anemia (1). Herbaspirillum species infections were hospital-onset in 5 patients and community-onset in 3. All symptomatic patients were treated with intravenous antibiotics, and their infections resolved. No environmental source or common mechanism of acquisition was identified. This is the first report of a hospital-based cluster of Herbaspirillum species infections. Herbaspirillum species are capable of causing bacteremia and sepsis in immunocompromised patients. Herbaspirillum species can be misidentified as Burkholderia cepacia by commercially available microbial identification systems.

Genetic analysis among environmental strains of Balamuthia mandrillaris recovered from an artificial lagoon and from soil in Sonora, Mexico

Since the first report of Balamuthia mandrillaris as a causative agent of granulomatous amoebic encephalitis in humans, the environmental niche of this amoeba was assumed to be restricted to soil and dust. A single isolation from water was recently made independently by us from Northern Mexico. Now we report the isolation of 8 new strains of B. mandrillaris from Mexico. This continues the pattern of an excess of isolates from North America, compared to other parts of the world. All of the new isolates are environmental isolates, 7 from water samples and one from soil. The identity of each isolate was confirmed by PCR and by examining the sequences of the mitochondrial 16S-like rRNA gene. Success in amplification was determined using comparisons of amplifications of DNA from the strain CDC: V039 and the water strain (ITSON-BM1) as positive controls. The DNA sequences of the new isolates were compared to older strains from clinical cases using phylogenetic analysis, showing very high sequence similarity. The similarity among the new isolates and with previous clinical and environmental isolates of B. mandrillaris was also examined using biochemical and immunological studies. High homogeneity of total protein products, and similarity in antigenic moiety among the eight new isolates and two controls was found. Taken together, the molecular and biochemical studies indicate very low levels of genetic variation within B. mandrillaris.

First Report of Diplodia seriata Causing Pear Branch Canker Dieback in California

California produces 26% of the United States pear crop on approximately 5,600 ha. A survey of seven northern California pear orchards (Pyrus communis cv. Bartlett) in summer 2010 revealed the presence of wedge-shaped cankers on 2- to 5-cm diameter branches, equating to 1- to 3-year-old wood. Many of the observed cankers occurred near pruning wounds, and there was decreased foliation on infected branches. Infected wood was surface disinfected with 95% ethanol and briefly flamed. After removing bark, small sections of diseased tissue were plated onto 4% potato dextrose agar (PDA) amended with 0.01% tetracycline and placed on the lab bench at 22°C until fungal growth emerged. Fungal colonies that were consistently isolated were transferred to fresh PDA using hyphal tip isolation. Fungal colonies were dark brown to gray with aerial mycelium and formed pycnidia after 15 days of incubation at 22°C. Conidia were brown, oval to oblong, and measured (16.5-) 20 to 24 (-26) × (7.5) 8.75 to 11 (-12.5) μm (n = 50). DNA from 14- to 21-day-old colonies was extracted and sequences of the rDNA internal transcribed spacer region and part of the β-tubulin gene were amplified using primers ITS4/ITS5 and Bt2a/Bt2b, respectively (2). The DNA sequences of fungal isolates from California showed 99 to 100% homology with the ex-type Diplodia seriata De Not. (1) CBS112555 deposited in GenBank. DNA sequences from three California isolates were submitted to GenBank with accession numbers KC937062, KC937065, KF481957, KF481598, KF481959, and KF481960. Pathogenicity tests were performed in March 2011 on 3-year-old Bartlett pear trees planted at an experimental farm in Davis, CA. A single, circular, 2-cm pruning wound at the top of the trunk was inoculated on each of three single-tree replications using 2-cm mycelial plugs from 14-day-old colonies growing on PDA. After inoculation, mycelial plugs were covered and sealed with Parafilm and aluminum foil for the duration of the trial. Three control trees were inoculated using sterile PDA plugs. Twelve months after inoculation, UCD103 and UCD105 were consistently re-isolated from the margin between necrotic and healthy tissue using the same methods described for the original isolation, and UCD102 was re-isolated in two out of three plants. The average lesion lengths of UCD102, UCD103, UCD105, and control plants were 12.5, 17.3, 23, and 1 mm, respectively. Control lesions were short and sterile, and seemed to be a physiological reaction from the plant. A second pathogenicity test was completed in 5 months beginning in June 2012. UCD105 was consistently re-isolated, and UCD102 and UCD103 were re-isolated in two out of three plants. The average lesion lengths for UCD102, UCD103, UCD105, and control plants were 2, 3, 5, and 1 mm, respectively. Compared to grapevine (Vitis vinifera), the pathogen grows more slowly in pear tissue under natural conditions. To our knowledge, this is the first report describing D. seriata as a causal agent of pear branch canker in California. Canker diseases can reduce the lifespan of perennial plants, ultimately leading to long term economic losses for growers (3).

Indy gene variation in natural populations confers fitness advantage and life span extension through transposon insertion

Natural selection acts to maximize reproductive fitness. However, antagonism between life span and reproductive success frequently poses a dilemma pitting the cost of fecundity against longevity. Here, we show that natural populations of Drosophila melanogaster harbor a Hoppel transposon insertion variant in the longevity gene Indy (I'm not dead yet), which confers both increased reproduction and longevity through metabolic changes. Heterozygosity for this natural long-lived variant has been maintained in isolates despite long-term inbreeding under laboratory conditions and advantageously confers increased fecundity. DNA sequences of variant chromosome isolates show evidence of selective sweep acting on the advantageous allele, suggesting that natural selection acts to maintain this variant. The transposon insertion also regulates Indy expression level, which has experimentally been shown to affect life span and fecundity. Thus, in the wild, evolution reaffirms that the mechanism of heterozygote advantage has acted upon the Indy gene to assure increased reproductive fitness and, coincidentally, longer life span through regulatory transposon mutagenesis.

Pseudonectria buxi causing leaf and stem blight on Buxus in Canada

An outbreak of a boxwood disease was observed in nurseries in southern Ontario in 2008, but the disease appears to have been present in Ontario for at least 15 years. From 2008 to 2010, over 300 fungal isolates were obtained from diseased samples, which included cuttings and whole plants. Almost half of the isolates (144) were found to belong to a single cultural morphotype which was confirmed as Pseudonectria buxi by DNA sequencing of several isolates. The remaining isolates were grouped into another seven major morphotypes, the largest of which accounted for 11 % of the isolates. Single representatives of all eight morphotypes were identified to species using morphological and molecular techniques, and were subjected to pathogenicity testing. Among them, only Pseudonectria buxi successfully satisfied Koch’s postulates on detached leaves, small twigs and rooted cuttings, which confirmed it as the causal agent of Volutella blight. Inoculation tests showed that wounds may be the major infection courts for P. buxi, since non-wounded inoculated tissues did not become diseased. Among several commonly grown boxwood cultivars in Ontario, ‘Green Gem’ was the most susceptible compared to ‘Green Velvet’, ‘Green Mound’, ‘Green Mountain’ or ‘Pincushion’, but all were found to be susceptible. Rather than behaving just as a saprophyte or secondary invader, P. buxi is capable of primary invasion after wounding, and causing extensive disease on live boxwood tissues.

Genomics and museum specimens

Nearly 25 years ago, Allan Wilson and colleagues isolated DNA sequences from museum specimens of kangaroo rats (Dipodomys panamintinus) and compared these sequences with those from freshly collected animals (Thomas et al. 1990). The museum specimens had been collected up to 78 years earlier, so the two samples provided a direct temporal comparison of patterns of genetic variation. This was not the first time DNA sequences had been isolated from preserved material, but it was the first time it had been carried out with a population sample. Population geneticists often try to make inferences about the influence of historical processes such as selection, drift, mutation and migration on patterns of genetic variation in the present. The work of Wilson and colleagues was important in part because it suggested a way in which population geneticists could actually study genetic change in natural populations through time, much the same way that experimentalists can do with artificial populations in the laboratory. Indeed, the work of Thomas et al. (1990) spawned dozens of studies in which museum specimens were used to compare historical and present-day genetic diversity (reviewed in Wandeler et al. 2007). All of these studies, however, were limited by the same fundamental problem: old DNA is degraded into short fragments. As a consequence, these studies mostly involved PCR amplification of short templates, usually short stretches of mitochondrial DNA or microsatellites. In this issue, Bi et al. (2013) report a breakthrough that should open the door to studies of genomic variation in museum specimens. They used target enrichment (exon capture) and next-generation (Illumina) sequencing to compare patterns of genetic variation in historic and present-day population samples of alpine chipmunks (Tamias alpinus) (Fig. 1). The historic samples came from specimens collected in 1915, so the temporal span of this comparison is nearly 100 years.

The Product of Nature Doctrine in the Myriad Saga II

In June 2013, the U.S. Supreme Court decided Association for Molecular Pathology et al. v. Myriad Genetics, Inc., et al., holding that “a naturally occurring DNA segment is a product of nature and not patent eligible merely because it has been isolated, but cDNA is patent eligible because it is not naturally occurring”.This case note gives an overview of the U.S. Supreme Court's decision, which is focused on the product of nature doctrine, and discusses its implications for the implementation of the criterion of isolation to DNA sequences and the United States Patent and Trademark Office's long-standing practice of granting patents on isolated DNA sequences (author's headnote).

First finding of Cryphonectria parasitica causing chestnut blight on Castanea sativa trees in England

Samples were collected from five affected trees and symptom-bearing bark pieces then placed in moist chambers at 18°C for up to 10 days to induce fungal sporulation. Cultures were made from spore masses extruding from the cankered bark onto potato dextrose agar (PDA). Isolations were also made onto PDA from the edge of necrotic lesions visible in the phloem tissue of cankered bark tissue. Fungal cultures were obtained from four of the five trees and the causal fungus was identified through morphological characteristics and DNA sequence data. DNA was extracted using the CTAB extraction protocol (Moller et al., 1992) and a portion of the ITS rDNA operon amplified and sequenced following the method of Gryzenhout et al., (2004). Resulting sequences were blasted against the nucleotide sequence database of the National Center for Biotechnology Information (NCBI) and closely allied sequences downloaded from NCBI and used to generate a DNA sequence phylogeny (Fig. 2). The DNA sequences of isolates collected in this study were very similar to many C. parasitica sequences found on GenBank including AY141859 (99% coverage, 99% identity) and JN942325 (100% coverage, 99% identity). This identification was corroborated by morphological characteristics of the isolated fungus which were the same as those described for Cryphonectria parasitica (Sivanesan & Holiday, 1981). Ascospores and asci of the C. parasitica isolates collected in England measured (7.5-)8-10(-12) x (3-)4(-5) μm and (32-)38-45(-48) x (5-)6-8(-9) μm respectively, which are in the range known for this species (Sivanesan & Holliday, 1981) (Fig. 3). Pathogenicity of C. parasitica was confirmed through Koch’s postulates. Mycelial plugs from three seven-day-old C. parasitica cultures were used to inoculate detached twigs of C. sativa, while controls used sterile PDA. Each inoculation was carried out in triplicate and inoculated twigs were incubated at 20°C for 5 weeks in a 9h/15 h light/dark cycle following which lesions were measured (Fig. 4). Cryphonectria parasitica was re-isolated from lesions confirming the pathogenicity of these isolates. This finding represents an example of incursion by an exotic pathogen into the UK and highlights the importance of plant health inspections and the difficulty of detecting infected plant material at the time of importation. Strategies are underway to manage and contain this outbreak while surveys are being conducted to determine if other sites have infected trees.

Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

BackgroundHawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required.FindingsDifferent bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants.ConclusionTo the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

DNA Aptamers against the Lup an 1 Food Allergen

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs.

English

  Mycoplasma synoviae (MS) is one of the important pathogens in chicken and turkey which cause great economic losses in poultry industry. M. synoviae has one serotype but there is heterogeneity among MS strains. The aim of this study was to analyze the DNA sequence of Mycoplasma synoviae isolates from Mazandran province poultry flocks of Iran and compare them with MS from other countries. For this purpose, 32 samples were collected from choanal cleft, trachea and air sac of flocks with respiratory symptom and cultured in specific PPLO medium. Ten out of the 32 samples were positive in culture and reacted with specific antisera. The 10 positive samples were subsequently used for molecular study. The PCR products containing 16s rRNA genome were generated with specific primers and then sequenced. Multiple alignment and phylogenic analysis of DNA sequences was performed with DNASTAR program. Alignment and comparison of the sequences in DANSTAR program revealed that 7 of 10 isolate are quite similar and 3 other isolate are different from group one. Phylogenic analysis of M. synoviae isolated from Mazandran province and 21 M. synoviae from the entire world revealed low homology among most of them. However, some of the Mazandran isolates showed high homology with M. synoviaeisolated from Brasilia. Overall, these results revealed the different molecular structure and heterogeneity among M. synoviae isolated from Mazandran province in this study.   Key words: Mycoplasma sinoviae, sequence, phylogenetic, poultry, Mazandran.

Pulmonary bacterial communities in surgically resected noncystic fibrosis bronchiectasis lungs are similar to those in cystic fibrosis.

Background. Recurrent bacterial infections play a key role in the pathogenesis of bronchiectasis, but conventional microbiologic methods may fail to identify pathogens in many cases. We characterized and compared the pulmonary bacterial communities of cystic fibrosis (CF) and non-CF bronchiectasis patients using a culture-independent molecular approach. Methods. Bacterial 16S rRNA gene libraries were constructed from lung tissue of 10 non-CF bronchiectasis and 21 CF patients, followed by DNA sequencing of isolates from each library. Community characteristics were analyzed and compared between the two groups. Results. A wide range of bacterial diversity was detected in both groups, with between 1 and 21 bacterial taxa found in each patient. Pseudomonas was the most common genus in both groups, comprising 49% of sequences detected and dominating numerically in 13 patients. Although Pseudomonas appeared to be dominant more often in CF patients than in non-CF patients, analysis of entire bacterial communities did not identify significant differences between these two groups. Conclusions. Our data indicate significant diversity in the pulmonary bacterial community of both CF and non-CF bronchiectasis patients and suggest that this community is similar in surgically resected lungs of CF and non-CF bronchiectasis patients.

New Phytophthoras in Western Australia’s natural ecosystems

Phytophthora spp. are recognised as important plant pathogens. Ten new and genetically diverse species, not previously reported from elsewhere, have recently been described from natural ecosystems in Western Australia (WA): Phytophthora multivora1; P. elongata; P. thermophila, P. gregata, P. gibbosa, P. litoralis; P. arenaria, P. constricta; P. fluvialis; and P. amnicola. They were identified by DNA sequencing of recent and historical isolates from the WA Department of Environment and Conservation (DEC). Several additional new taxa await description. New records for WA of at least eight other Phytophthora taxa that are known overseas, some of them as yet undescribed, have also been confirmed: P. inundata; P. taxon niederhauserii, P. taxon asparagi, P. taxon PgChlamydo, P. taxon personii; P. taxon salixsoil; P. palmivora and P. rosacearum. Furthermore, numerous Phytophthora hybrids have been identified in natural vegetation and waterways in WA. The phylogenetic relationships of the new WA Phytophthora taxa, with their nearest relatives, are shown in Figure 1.

Ribosomal DNA Sequence Based Characterization of Trichoderma Isolates Antagonistic to Colletotrichum falcatum Causing Red Rot Disease of Sugarcane

Trichoderma bioagent isolates cultured from the sugarcane rhizosphere of subtropical India, and established efficient against Colletotrichum falcatum Went (red rot pathogen) in sugarcane are described based on phylogenetic analysis of nucleotide sequences. DNA sequences of the isolates included the internal transcribed spacer (ITS) regions of the rDNA cluster (ITS1-5.8S- ITS2). The 5.8S region was found conserved and much of the sequence variability was due to indels or transition/transversion mutations in the ITS1 and 2 regions. Both the isolates belonged to the T. harzianum clade. These isolates are distinct yet have close genetic similarity with several established strains/isolates of T. harzianum reported worldwide. Correct identification of these bioagents would augment the effective utilization of these fungi of immense agricultural importance in managing the red rot disease of sugarcane.

DisseminatedNocardia cyriacigeorgicaPresenting as Right Lung Abscess and Skin Nodule

Nocardia species are ubiquitous in the environment and can be found worldwide. Direct inhalation remains the most commonly attributed route of infection with Nocardia asteroides complex, causing 50% of invasive infections. Improved molecular methods have identified a significant proportion of N. asteroides complex isolates to be Nocardia cyriacigeorgica. We report a case of a 58-year-old male working as a contractor in Bagram Air Force Base, Afghanistan, with disseminated N. cyriacigeorgica involving the lung, brain, and dermis. Diagnosis was facilitated by early identification of branched, filamentous bacteria using Fite and gram staining along secA DNA sequencing of clinical isolates. Our patient is the first confirmed case of N. cyriacigeorgica infection in Afghanistan.

Molecular Characterization of Tomato Leaf Curl Hainan Virus, a New Begomovirus, and Evidence for Recombination

AbstractVirus isolates HaNHK7 and HaNHK8 were obtained from tomato plants showing leaf curl symptoms in Hainan Province of China. The complete DNA sequences of both isolates were determined to be 2748 nucleotides, with all the characteristic features of begomovirus genome organization. The two isolates share 99.8% identity with each other but have <88.3% nucleotide sequence identity with other begomoviruses. Consequently, HaNHK7 and HaNHK8 are considered to be isolates of a novel Begomovirus species, for which the name Tomato leaf curl Hainan virus (ToLCHnV) is proposed. ToLCHnV is most closely related to Papaya leaf curl China virus (PaLCuCNV, AJ558117) (88.3% in total nucleotide sequences). However, the AC2 gene more resembles that of Ageratum leaf curl virus (ALCuV, AJ851005) and AC1 and AC4 genes resemble those of Tomato leaf curl Vietnam virus (ToLCVV, AF264063). Sequence analyses suggest that ToLCHnV may have arisen by recombination between viruses related to PaLCuCNV, ALCuV and ToLCVV. Neither the DNA‐B component nor the DNA‐β molecule was found to be associated with ToLCHnV isolates.

Inhibition of Human Immunodeficiency Virus Type 1 by Triciribine Involves the Accessory Protein Nef

Triciribine (TCN) is a tricyclic nucleoside that inhibits human immunodeficiency virus type 1 (HIV-1) replication by a unique mechanism not involving the inhibition of enzymes directly involved in viral replication. This activity requires the phosphorylation of TCN to its 5' monophosphate by intracellular adenosine kinase. New testing with a panel of HIV and simian immunodeficiency virus isolates, including low-passage-number clinical isolates and selected subgroups of HIV-1, multidrug resistant HIV-1, and HIV-2, has demonstrated that TCN has broad antiretroviral activity. It was active in cell lines chronically infected with HIV-1 in which the provirus was integrated into chromosomal DNA, thereby indicating that TCN inhibits a late process in virus replication. The selection of TCN-resistant HIV-1 isolates resulted in up to a 750-fold increase in the level of resistance to the drug. DNA sequence analysis of highly resistant isolate HIV-1(H10) found five point mutations in the HIV-1 gene nef, resulting in five different amino acid changes. DNA sequencing of the other TCN-resistant isolates identified at least one and up to three of the same mutations observed in isolate HIV-1(H10). Transfer of the mutations from TCN-resistant isolate HIV-1(H10) to wild-type virus and subsequent viral growth experiments with increasing concentrations of TCN demonstrated resistance to the drug. We conclude that TCN is a late-phase inhibitor of HIV-1 replication and that mutations in nef are necessary and sufficient for TCN resistance.

Assessment of DNA Damage by RAPD inParacentrotus lividusEmbryos Exposed to Amniotic Fluid from Residents Living Close to Waste Landfill Sites

The aim of this study was to assess the genotoxic effects of environmental chemicals on residents living near landfills. The study was based on samples of amniotic fluid from women living in the intensely polluted areas around the Campania region of Italy compared to a nonexposed control group. We evaluated the genetic effects that this amniotic fluids collected in contaminated sites had on Paracentrotus lividus embryos. DNA damage was detected through changes in RAPD (Random Amplified Polymorphism DNA) profiles. The absence of the amplified DNA fragments indicated deletions in Paracentrotus lividus DNA exposed to the contaminated amniotic fluids when compared to equal exposure to uncontaminated fluids. These results show the ability of RAPD-PCR to detect and isolate DNA sequences representing genetic alterations induced in P. lividus embryos. Using this method, we identified two candidate target regions for DNA alterations in the genome of P. lividus. Our research indicates that RAPD-PCR in P. lividus embryo DNA can provide a molecular approach for studying DNA damage from pollutants that can impact human health. To our knowledge, this is the first time that assessment of DNA damage in P. lividus embryos has been tested using the RAPD strategy after exposure to amniotic fluid from residents near waste landfill sites.

Genome Analysis of Multi- and Extensively-Drug-Resistant Tuberculosis from KwaZulu-Natal, South Africa

The KZN strain family of Mycobacterium tuberculosis is a highly virulent strain endemic to the KwaZulu-Natal region of South Africa, which has recently experienced an outbreak of extensively-drug resistant tuberculosis. To investigate the causes and evolution of drug-resistance, we determined the DNA sequences of several clinical isolates - one drug-susceptible, one multi-drug resistant, and nine extensively drug-resistant - using whole-genome sequencing. Analysis of polymorphisms among the strains is consistent with the drug-susceptibility profiles, in that well-known mutations are observed that are correlated with resistance to isoniazid, rifampicin, kanamycin, ofloxacin, ethambutol, and pyrazinamide. However, the mutations responsible for rifampicin resistance in rpoB and pyrazinamide in pncA are in different nucleotide positions in the multi-drug-resistant and extensively drug-resistant strains, clearly showing that they acquired these mutations independently, and that the XDR strain could not have evolved directly from the MDR strain (though it could have arisen from another similar MDR strain). Sequencing of eight additional XDR strains from other areas of KwaZulu-Natal shows that they have identical drug resistant mutations to the first one sequenced, including the same polymorphisms at sites associated with drug resistance, supporting the theory that this represents a case of clonal expansion.