Distinct Banding Patterns Research Articles

Restriction Fragment Length Polymorphism Genotyping of Human Staphylococcus aureus Isolates From Two Hospitals in Urmia Region of Iran Using the coa Gene

: Background: Staphylococcus aureus has become an emerging public health concern. Markers that differentiate tissue-specific lineages are needed to trace the sources of strains.Objectives: The aims of this study were to determine the genotypic characteristics ofS. aureus isolates that are associated with skin and urinary tract infections using polymorphisms in the coagulase gene.Materials and Methods: Coagulase gene variants among 26 S. aureus isolates from human infected skin (n = 10) and urine (n = 16) samples were investigated by amplification of the repeat units encoding the hypervariable region of the coagulase gene. The amplicons ranged from 490-790 bp and were subjected to restriction fragment length polymorphism (RFLp) analysis with HaeIII.Results: In total, 6 distinct RFLp banding patterns were observed, designated C1-C6.The C1 pattern predominated in skin and urine isolates. Notably, the C3, C5, and C6 patterns were present in isolates from urine, whereas the C2 and C4 genotypes were preferentially detected in skin sample isolates.Conclusions: These data demonstrate the widespread prevalence of certain genotypes and tissue-specific tendency of other genotypes, suggesting the existence of lineage- and tissue-specific genes that mediate the development of tissue-specific pathogenicities of S. aureus isolates. Implication for health policy/practice/research/medical education:Results of the current study also showed that the repeat region of coa gene can be useful for typing and grouping of skin and urinary tract associated S. aureus isolates. Please cite this paper as:Talebi-Satlou R, Ahmadi M, Dastmalchi Saei H. Restriction Fragment Length Polymorphism Genotyping of Human Staphylococcus aureus Isolates From Two Hospitals in Urmia Region of Iran Using the coa Gene. Jundishapur J Microbiol.2012;5(2):416-20. DOI: 10.5812/jjm.3522 Copyright © 2012 Kowsar Corp. All rights reserved.

Rapid Identification of Sporothrix Species by T3B Fingerprinting

This article describes PCR fingerprinting using the universal primer T3B to distinguish among species of the Sporothrix complex, S. brasiliensis, S. globosa, S. mexicana, and S. schenckii. This methodology generated distinct banding patterns, allowing the correct identification of all 35 clinical isolates at the species level, confirmed by partial calmodulin (CAL) gene sequence analyses. This methodology is simple, reliable, rapid, and cheap, making it an ideal routine identification system for clinical mycology laboratories.

A Set of SCAR Markers Efficiently Differentiating Hybrid Rice

Molecular markers have been widely used in crop genetic improvement, seed test and genetic mapping. Of which, sequence characterized amplified region (SCAR) markers are particularly popular for its diversity, stable reproducibility, and suitability for analyzing large number of samples. In this study, 500 random amplified polymorphic DNA (RAPD) primers were tested, and a set of SCAR markers comprising 37 pairs of loci-specific primers were developed from the DNA fragments ranging from 300 to 1000 bp which correspond to the stable, distinctive RAPD banding patterns. Using these SCAR markers, 59 hybrid rice combinations were assessed and distinguished into 58 subgroups at the similarity coefficient of 0.97 in a genetic clustering tree based on the allele diversities of the SCAR markers. Furthermore, 13 hybrid rice combinations were reassayed with 40 randomly selected simple sequence repeat (SSR) markers to evaluate the effectiveness of these SCAR markers. SSR markers produced similar results to SCAR markers as the 13 hybrid rice combinations were completely separated at the similarity coefficient of 0.91 in the clustering tree established from SSR patterns. Taken together, SCAR markers prove to be effective tools for identifying and differentiating hybrid rice combinations.

Genetic Variability within the Population of the Vulnerable Mindanao Endemic Blue-Capped Kingfisher (Actenoides hombroni) Using Polymorphic DNA Markers

The restricted range and small population size of Mindanao endemic blue-capped kingfisher ( Actenoides hombroni ) and the degradation of its habitat provide an ideal case to study the genetics of this species. DNA samples were taken from A. hombroni netted from Nursery and Gabunan, Rogongon, Iligan City and Kimangkil, Bukidnon. Random amplified polymorphic DNA (RAPD) analysis and agarose gel electrophoresis was used to assess the genetic variability within its population. The random primer used had generated 25 reproducible RAPD markers showing three distinct banding patterns with low polymorphism. Shared or specific fragments were counted and genetic similarity within population was calculated. The similarity coefficient (F) estimation revealed that there were more pairs of individuals with F values equal to 1 (high genetic similarity) and having no F value equal to 0 (low genetic similarity). Results suggested that there is a considerable low genetic diversity that existed within the population of A. hombroni, which could be explained by their geographic proximity. Findings of this study are significant most specially in the formulation of conservation strategies for the Mindanao endemic and vulnerable A. hombroni. Keywords - Actenoides hombroni, genetic variability, PCR, RAPD markers, similarity coefficient, Rogongon, Iligan City, Mt. Kimangkil, Bukidnon

Effects of Culture Conditions on Growth and Biochemical Profile of Chlorella Vulgaris

The effects of culture conditions at different temperature and light regimes on growth and the contents of chlorophyll-a, chlorophyll-b, total carotenoids, total protein and total free amino acids of Chlorella vulgaris were determined. The growth of C. vulgaris in terms of optical density (0.42 at 670 nm), cell count (440 x 104 cells/ml) and dry weight (30.2 mg/50 ml), and the amount of chlorophyll- a (2.16%), chlorophyll- b (0.59%) and total protein, was found higher at the temperature 25-30°C and natural day light receiving through the north facing window of the growth room. Although, the amount of total carotenoids (0.440%) and free amino acids (834 μg/gm fresh weight) were found maximum in continuous light at 30-35°C, not much differences in the amount of carotenoids (0.385%) and free amino acids (822 μg/gm fresh weight) were found at 25-30°C and natural day light. The natural day light at 25-30°C was also proved proficient, as distinct banding pattern with unique polypeptides such as 15KDa, 47KDa and 50KDa, on the other hand, 23KDa, 26KDa and 36KDa appeared in all samples, these bands were not affected by light and temperature. Our results indicate that among all five culture conditions tested, the cultures kept at north facing window receiving natural day light at temperature 25-30°C, show best growth and higher contents of biochemicals that will be beneficial to use Chlorella for high nutritive purpose.

Cytogenetical and Molecular Characterization of Five Commercial Varieties in <i>Trichosanthes anguina</i> L.

Five commercial varieties of Trichosanthes anguina (Snake Gourd), namely Turag, Dhaka Green, Super Long Green, Anika and Apurbo, were investigated cytogenetically and at the molecular level for authentic characterization. The variety Super Long Green was found to possess 2n=23 chromosomes and considered as a primary trisomic. 2n=22 chromosomes were found in the other 4 varieties. The total length of the 2n chromosome complement was largest in Anika and smallest in Apurbo. Only one extra large chromosome was found in Turag and Anika suggesting it had either been transferred from different genome or was a case of repeated duplication. No decrease in chromosomal length was found in Turag, a moderate decrease was found in Dhaka Green and Anika, and a sharp decrease in Super Long Green and Apurbo. A pair of satellites was found in Turag. Only a stain specific satellite was found in Dhaka Green. No CMA band was found in Turag and Super Long Green. The other 3 varieties showed distinct CMA banding pattern in respect of number, size, location and percentage of GC-rich area. The 5 varieties showed distinct RAPD bands with 6 different primer combinations. In addition, the Apurbo and Turag varieties showed unique DNA fragments with the primer combinations OPA-3 and OPA-4 respectively. A combination of cluster and karyotytpe analysis clearly indicated that Turag and Apurbo were distinctly related from the other 3 varieties. Therefore, with the help of cytogenetical and RAPD analysis, it was possible to characterize each variety.

Characterization of Aeromonas hydrophila through RAPD-PCR and SDS-PAGE Analysis

Aeromonas hydrophila isolated from fish (Labeo rohita), pond water, river water, raw meat of chicken and mutton and raw cow milk were characterized through Random Amplified Polymorphic DNA (RAPD) analysis and Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) of cellular proteins. RAPD-PCR amplification of bacterial DNA was done by using ten random primers (OPA-01 to 10) and found some distinct banding pattern on agarose gel. RAPD profile was studied with each isolate and absolute polymorphism indicating its application as an ideal tool for molecular characterization. Other methods like morphological, serological and biochemical characterization gives contradictory results and total cellular protein profiling does not show any significant polymorphism for identification and discrimination.

A pathogenic Vibrio harveyi lineage causes recurrent disease outbreaks in cultured Japanese flounder ( Paralichthys olivaceus) and induces apoptosis in host cells

Vibrio harveyi is an important aquaculture pathogen that can infect a large number of marine animals. In this study, we examined three V. harveyi strains (VJ1, VJ2, and VJ3) isolated in the years of 2008, 2009, and 2010 respectively from diseased Japanese flounder ( Paralichthys olivaceus) in a fish farm in China that had been inflicted with disease outbreaks. VJ1, VJ2, and VJ3 exhibited comparable virulence in a flounder model and induced symptoms in experimentally infected fish that are indistinguishable from those of naturally diseased fish. 16S rDNA and multilocus sequence analysis indicated that VJ1, VJ2, and VJ3 share 98–100% sequence identities with known V. harveyi isolates in 16S rDNA and four housekeeping genes. VJ1, VJ2, and VJ3 have distinct and identical AFLP banding patterns, which differ profoundly from those of two environmental V. harveyi isolates. VJ1, VJ2, and VJ3 exhibited comparable growth profiles and hemolytic activities and possess the vhh hemolysin gene. Compared to an environmental isolate, VJ1, VJ2, and VJ3 showed comparable and significantly stronger resistances against the bactericidal effect of host serum and induced higher levels of respiratory burst activity in head kidney macrophages. However, VJ1, VJ2, and VJ3 were unable to proliferate in macrophages. When incubated with a cultured cell line of flounder, VJ1, VJ2, and VJ3 induced apoptosis characterized by the occurrence of DNA fragmentation, apoptotic bodies, and elevated caspase 3 activity. Taken together, these results indicate that VJ1, VJ2, and VJ3 are probably clones of the same V. harveyi lineage that has persisted during the examined three-year time frame and likely caused recurrent disease outbreaks via a virulence mechanism involving serum resistance and triggering apoptosis in host cells.

Molecular, physiological and pathogenic variability of Pyrenochaeta lycopersici associated with corky rot disease of tomato plants in Turkey

Isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot on tomato plants, were assessed for physiological and genetic characteristics using conventional and molecular techniques. All isolates were able to produce necrosis on tomato roots and classified into temperature group according to the optimal growth temperatures. Specific-PCR assays and DNA sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer region confirmed the existence of both types (Type 1 and Type 2) of the pathogen among the isolates tested. All isolates were identified as Type 2 except for isolate Pl-4, which was classified as Type 1. Restriction fragment length polymorphism (RFLP) analysis with six enzymes resulted in three distinct banding patterns among the isolates depending on the length and restriction profiles of the rDNA intergenic spacer region. Inter-simple sequence-repeat analysis revealed a high level of genetic diversity among the isolates in agreement with the data of RFLP analysis. These results indicated that there were three different intraspecific groups among Turkish isolates of P. lycopersici. The presented study is the first attempting to characterize Turkish isolates of P. lycopersici. The results obtained will be useful in screening of tomato seedlings for resistance to P. lycopersici.

Cellular prion proteins in human platelets show a phenotype different to those in brain tissues

Prion diseases are characterized by high accumulation of infectious prion proteins (PrP(Sc)) in brains. PrP(Sc) are propagated by the conversion of host-encoded cellular prion proteins (PrP(C)) which are essential for developing the disease but are heterogeneously expressed in brains. The disease can be transmitted to humans and animals through blood and blood products, however, little attention has been given to molecular characterization of PrP(C) in blood cells. In this presented study, we characterized phenotypically PrP(C) of platelets (plt) and characterized the proteins regarding their glycobanding profiles by quantitative immunoblotting using a panel of monoclonal antibodies. The glycosylation patterns of plt and brain PrP(C) were compared using the ratios of di-, mono-, and non-glycosylated prions. The detergent solubility of plt and brain PrP(C) was also analyzed. The distinct banding patterns and detergent solubility of plt PrP(C) differed clearly from the glycosylation profiles and solubility characteristics of brain PrP(C). Plt PrP(C) exhibited single or only few prion protein types, whereas brain PrP(C) showed more extensive banding patterns and lower detergent solubility. Plt PrP(C) are post-translational modified differently from PrP(C) in brain. These findings suggest other or less physiological functions of plt PrP(C) than in brain.

ISSR Analysis of Somaclonal Variation in Callus-Derived Plants of Amorphophallus Rivieri Durieu

This communication reports detection of somaclonal variation among tissue culture-raised plants of Amorphophallus rivieri Durieu, an economically important crop in China, with high content of glucomannan in its corms. A population of regenerated plants was obtained from a single donor plant of A. rivieri via corm organogenesis, and 28 plants were randomly selected as a representative sample and subjected to analysis of somaclonal variation using inter-simple sequence repeat (ISSR) markers. Of the 26 ISSR primers screened, 13 gave distinct and reproducible band patterns, yielding 131 bands with an average of 10.1 bands per primer. Ten primers were polymorphic and generated 16 polymorphic bands with 12.2% mean polymorphism. Based on the ISSR data from the regenerated plants and the donor plant, Jaccard's similarity coefficients were calculated; they ranged from 0.961 to 1.000 with a mean of 0.982. A dendrogram was constructed using the unweighted pair group method with arithmetic mean (UPGMA); it showed that a majority of regenerated plants (including the donor plant) clustered closely, with a mean similarity coefficient of 0.987. Low somaclonal variation observed in the regenerated plants indicates that rapid propagation of A. rivieri via corm organogenesis is a practicable method with a low risk of genetic instability.

Genetic variation among species, races, forms and inbred lines of lac insects belonging to the genus Kerria (Homoptera, Tachardiidae)

The lac insects (Homoptera: Tachardiidae), belonging to the genus Kerria, are commercially exploited for the production of lac. Kerria lacca is the most commonly used species in India. RAPD markers were used for assessing genetic variation in forty-eight lines of Kerria, especially among geographic races, infrasubspecific forms, cultivated lines, inbred lines, etc., of K. lacca. In the 48 lines studied, the 26 RAPD primers generated 173 loci, showing 97.7% polymorphism. By using neighbor-joining, the dendrogram generated from the similarity matrix resolved the lines into basically two clusters and outgroups. The major cluster, comprising 32 lines, included mainly cultivated lines of the rangeeni form, geographic races and inbred lines of K. lacca. The second cluster consisted of eight lines of K. lacca, seven of the kusmi form and one of the rangeeni from the southern state of Karnataka. The remaining eight lines formed a series of outgroups, this including a group of three yellow mutant lines of K. lacca and other species of the Kerria studied, among others. Color mutants always showed distinctive banding patterns compared to their wild-type counterparts from the same population. This study also adds support to the current status of kusmi and rangeeni, as infraspecific forms of K. lacca.

Sylvatic and peridomestic populations of Triatoma pseudomaculata are not significantly structured by habitat, as revealed by two genetic markers

Chagas disease remains a public health concern in Brazil and other Latin American countries, mainly due to the potential domiciliation of native triatomine species. We analyzed the genetic variability of Triatoma pseudomaculata in sylvatic and peridomestic ecotopes throughout three localities in the northeastern state of Bahia, Brazil. We studied polymorphisms generated by random amplified polymorphic DNA (RAPD) and isoenzyme electrophoresis analyses. Based on RAPD analysis, each specimen was assigned to one of three genetic clusters. Although all sylvatic specimens from one locality were grouped into the same cluster, sylvatic and peridomestic specimens from the other two localities were broadly distributed between the remaining two clusters, suggesting that geographic population structuring was not occurring. Furthermore, isoenzyme analysis suggested that distinct populations were in Hardy-Weinberg equilibrium. Low statistical values for Wright's Fst index also supported the absence of population structuring and suggested the occurrence of panmixia. We conclude that genetic flow occurs between sylvatic and peridomestic T. pseudomaculata populations, probably as a consequence of passive and active dispersion of the insects, associated with deforestation and anthropic transformations.

Detection of genomic changes in transgenic Bt rice populations through genetic fingerprinting using amplified fragment length polymorphism (AFLP)

A highly precise molecular marker technique, Amplified Fragment Length Polymorphism (AFLP) was used to characterize the changes in genomic structure occurred due to introduction of foreign gene and/ or due to the induced tissue culture stress during the development of various transgenic Bt rice populations. The transgenic rice plants having complex to simple Bt gene and selectable marker gene organizations were selected from the six insect resistant Bt rice lines. Our results clearly demonstrate that integration pattern, extent of rearrangement of foreign DNA and method of transformation may influence the genomic changes in transgenic rice populations. Eleven of fourteen AFLP primer combinations tested, generated distinct scorable banding pattern, which were further used in this study. Three primer combinations E-TA / M-CTT (IRRI-NPT), E-AG / M-CAC (Tulasi) and E-AA / M-CAG (IR 68899B) produced only monomorphic bands in all the transgenic and control rice plants .A percentage of 0.61% of 430 million bases of haploid rice genome were examined by the use of 11 AFLP primer combinations employed in this study. The DNA fingerprints generated by AFLP analysis, of each Bt rice population was compared with their respective non-transgenic control and was found to be > 97% similar. This indicated that the introduction of Bt gene into the genome of six rice varieties showed few genomic changes. The comparison shows that fewer changes were observed among the transgenic plants developed by Agrobacterium infection than that of developed through particle bombardment. Transgene integration pattern and their copy number were associated with the extent of genomic changes observed in the transgenic Bt rice varieties. AFLP fingerprints of the six transgenic rice varieties evidenced few, but consistent polymorphic bands between the transgenic individuals with different PI values. Relationships among the transgenic populations with their control rice plants were expressed in the form of dendrograms.

Molecular differentiation of the species of two squid families ( Loliginidae and Ommastrephidae) based on a PCR study of the 5S rDNA gene

The present study aimed to demonstrate the effectiveness of the 5s rDNA gene for the identification of commercially-valuable species of cephalopod belonging to the families Loliginidae and Ommastrephidae. Our results demonstrate distinct banding patterns in each of the six species sampled ( Loligo surinamensis; Loligo sanpaulensis; Lolliguncula brevis; Sepiotheuthis sepioidea; Ornithoteuthis antillarum; Illex argentinus), as well as diagnostic traits at the genus and probably family levels. The results emphasize the efficiency of the 5s rDNA marker as a low-cost and rapid forensic technique, which not only permits the identification of species, but also differentiation of members of the Loliginidae and Ommastrephidae.

USE OF MOLECULAR MARKERS IN CROP IMPROVEMENT OF PUNICA GRANATUM L. FOR WATER STRESS TOLERANCE

Pomegranate (Punica granatum L.) is an important commercial fruit crop of India. The genetics of pomegranate differs in many aspects from that of seasonal crops due to many biological limitations. Perennial and heterozygous nature of pomegranate precludes the development of inbred lines, near isogenic lines (NIL), recombinant inbred lines (RIL) and back cross progenies. Hence, the novel techniques like molecular markers can be employed for improvement of pomegranate. There are two different types of molecular markers in use and in this study PCR based marker, i.e., Randomly Amplified Polymorphic DNA (RAPD) were used. The DNA finger prints used to correlate the water stress tolerance based on relative water content (RWC). Bulk segregant analysis (BSA) was used in 124 F 2 mapping population which was raised by crossing genetically diverse parents for the water stress tolerance traits viz., crop canopy air temperature difference, specific leaf weight, RWC and carbon isotope discrimination (Δ). The bulks and parents were compared using finger prints and no distinct banding pattern was observed. The results of this investigation could be used as preliminary study in improvement of fruit crops.

Nuclear protein matrix in giant nuclei from salivary glands of Chironomus plumosus

Polythene chromosomes from salivary glands of Chironomus plumosus were treated in situ in order to reveal residual nuclear protein matrix (NPM). It was shown that after the removal of H1-histones by 0.6 M NaCl the general morphology of chromosomes is preserved, revealing distinct banding pattern. Further treatment of chromosomes with 2 M NaCl and DNase completely disorganized the structure of chromosome bodies and patterns of banding. Preliminary treatment of salivary glands with 2 mM CuCl2 resulted in stabilization of the structure of polythene chromosome in every stage of histone and DNA extractions. Stabilized chromosomes maintained their morphology and banding patterns observed by phase contrast or after the staining with Brilliant blue. Thus, after the removal of histones and DNA, stabilized chromosomes retain their morphological features, which depend on the presence of NMP. In stabilized polythene chromosomes, in spite of the absence of histones and DNA, topoisomerase IIα retains its localization, typical for untreated chromosomes.

Diversity and Activity of PAH-Degrading Bacteria in the Phyllosphere of Ornamental Plants

Phyllosphere bacteria on ornamental plants were characterized based on their diversity and activity towards the removal of polycyclic aromatic hydrocarbons (PAHs), the major air pollutants in urban area. The amounts of PAH-degrading bacteria were about 1-10% of the total heterotrophic phyllosphere populations and consisted of diverse bacterial species such as Acinetobacter, Pseudomonas, Pseudoxanthomonas, Mycobacterium, and uncultured bacteria. Bacterial community structures analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis from each plant species showed distinct band patterns. The uniqueness of these phyllosphere bacterial communities was partly due to the variation in leaf morphology and chemical properties of ornamental plants. The PAH degradation activity of these bacteria was monitored in gas-tight systems containing sterilized or unsterilized leaves. The results indicated that phyllosphere bacteria on unsterilized leaves were able to enhance the activity of leaves for phenanthrene removal. When compared between plant species, phenanthrene removal efficiency corresponded to the size of phenanthrene-degrading bacteria. In addition, phyllosphere bacteria on Wrightia religiosa were able to reduce other PAHs such as acenaphthylene, acenaphthene, and fluorine in 60-ml glass vials and in a 14-l glass chamber. Thus, phyllosphere bacteria on ornamental plants may play an important role in natural attenuation of airborne PAHs in urban areas.

An efficient method for DNA extraction from Cladosporioid fungi

We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A(260)/A(280) ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyses.

Comparative RAPD analysis and pollen structure studies of Bellis perennis L.

Random amplified polymorphic DNA (RAPD) is one of the easiest and most commonly used molecular techniques for genetic variability analysis. In the present study, the genetic diversity of analysis of Bellis perennis L. collected in 4 Turkish geographical locations was investigated by RAPD-PCR analysis. In addition to RAPD, pollen analysis of the 4 plant populations was also conducted. Out of 40 RAPD primers tested, 12 exhibited distinct banding patterns showing 45% to 79% polymorphism. Plants from these 4 localities were almost the same morphologically. Considerable differences were observed in the RAPD profile and pollen shape of plants from the Rize/Dereköy location. The results of this study clearly show that there are significant variations in the field collected populations of B. perennis from different geographic and climatic locations in Turkey. Environmental conditions may help us to determine the genomic structures of species.