Digestion Of Total DNA Research Articles

Isolation of a Pericentromeric Satellite DNA Family in Chnootriba argus (Henosepilachna argus) with an Unusual Short Repeat Unit (TTAAAA) for Beetles

Ladybird beetles (Coccinellidae) are one of the largest groups of beetles. Among them, some species are of economic interest since they can act as a biological control for some agricultural pests whereas other species are phytophagous and can damage crops. Chnootriba argus (Coccinellidae, Epilachnini) has large heterochromatic pericentromeric blocks on all chromosomes, including both sexual chromosomes. Classical digestion of total genomic DNA using restriction endonucleases failed to find the satellite DNA located on these heterochromatic regions. Cloning of C0t-1 DNA resulted in the isolation of a repetitive DNA with a repeat unit of six base pairs, TTAAAA. The amount of TTAAAA repeat in the C. argus genome was about 20%. Fluorescence in situ hybridization (FISH) analysis and digestion of chromosomes with the endonuclease Tru9I revealed that this repetitive DNA could be considered as the putative pericentromeric satellite DNA (satDNA) in this species. The presence of this satellite DNA was tested in other species of the tribe Epilachnini and it is also present in Epilachna paenulata. In both species, the TTAAAA repeat seems to be the main satellite DNA and it is located on the pericentromeric region on all chromosomes. The size of this satDNA, which has only six base pairs is unusual in Coleoptera satellite DNAs, where satDNAs usually have repeat units of a much larger size. Southern hybridization and FISH proved that this satDNA is conserved in some Epilachnini species but not in others. This result is in concordance with the controversial phylogenetic relationships among the genera of the tribe Epilachnini, where the limits between genera are unclear.

Pulsed-Field Gel Electrophoresis: A Tool for Molecular Epidemiology of Bacteria

Pulsed-field gel electrophoresis (PFGE) is the most widely used technique for epidemiological and molecular biology investigations to make long-range physical maps of prokaryotic and eukaryotic genomes. PFGE is a unified typing system for epidemiological investigations of most bacterial pathogens. These methods have been used and adopted for several medically important bacterial species, but have had limited application for veterinary pathogens. Through this technique, we can investigate the geographical comparisons, heterogeneity examination, epidemiology and outbreaks. Accurate epidemiologic investigations require an assessment of relatedness between individuals with similar infections in order to determine if animal to animal spread has occurred. Epidemiologic typing systems are used to study population dynamics and spread of organisms that undergo clonal reproduction (i.e. bacteria). The digestion of total genomic DNA is done with restriction endonucleases. These enzymes cut the chromosome at infrequent intervals, producing relatively large fragments that can then be separated by using PFGE. Typing systems must be assessed according to performance criteria, namely, typeability, reproducibility, stability, discriminatory power, epidemiologic concordance and typing system concordance.

Strain typing of acetic acid bacteria responsible for vinegar production by the submerged elaboration method

Strain typing of 103 acetic acid bacteria isolates from vinegars elaborated by the submerged method from ciders, wines and spirit ethanol, was carried on in this study. Two different molecular methods were utilised: pulsed field gel electrophoresis (PFGE) of total DNA digests with a number of restriction enzymes, and enterobacterial repetitive intergenic consensus (ERIC) – PCR analysis. The comparative study of both methods showed that restriction fragment PFGE of SpeI digests of total DNA was a suitable method for strain typing and for determining which strains were present in vinegar fermentations. Results showed that strains of the species Gluconacetobacter europaeus were the most frequent leader strains of fermentations by the submerged method in the studied vinegars, and among them strain R1 was the predominant one. Results showed as well that mixed populations (at least two different strains) occurred in vinegars from cider and wine, whereas unique strains were found in spirit vinegars, which offered the most stressing conditions for bacterial growth.

Molecular Characterization of Size and Copy Number of the Human Insulin Gene in Transfected Rat Islet Tumor Cell Clones

Two stable transfectants, NHI-6F and NHI-5B, obtained by cotransfection of the human insulin gene (a 15 kb EcoRI genomic fragment) with pSV2-neo into MSL-cells (pluripotent rat islet tumor cells), were characterized with regard to copy number and size of the inserted human sequences by Southern blot analyses using appropriate restriction enzymes. Digestion of total genomic DNA from the NHI-6F clone with EcoRI, SacI and BglII gave in all cases single human insulin gene fragments of 25 kb, 28 kb and 11 kb, respectively. From these data we were able to conclude that NHI-6F carries a single copy of the human insulin gene and that this copy has at least retained the 5' BglI and the 3' BglII flanking sites of human origin. Digestion of NHI-5B DNA gave a different banding pattern compared to NHI-6F (EcoRI: 20 kb; SacI: 7.8 kb; BglII: 11 kb). These data indicate the presence of a single human insulin gene copy in clone NHI-5B with the preservation of the 5' SacI and 3' BglII human flanking sites. In conclusion, the two transfected rat islet tumor clones, NHI-6F and -5B, carry a single copy of the human insulin gene. Each copy is presumably functionally active since known promoter and enhancer elements reside within 400 bp 5' to the gene. NHI-6F and -5B carry 2,500 bp and 4,500 bp 5' to the gene, respectively.

Molecular and cytogenetic characterization of an AT-rich satellite DNA family in Urvillea chacoensis Hunz. (Paullinieae, Sapindaceae)

Urvillea chacoensis is a climber with 2n = 22 and some terminal AT-rich heterochromatin blocks that differentiate it from other species of the genus. The AT-rich highly repeated satellite DNA was isolated from U. chacoensis by the digestion of total nuclear DNA with HindIII and XbaI and cloned in Escherichia coli. Satellite DNA structure and chromosomal distribution were investigated. DNA sequencing revealed that the repeat length of satDNA ranges between 721 and 728 bp, the percentage of AT-base pairs was about 72-73% and the studied clones showed an identity of 92.5-95.9%. Although this monomer has a tetranucleosomal size, direct imperfect repetitions of ~180 bp subdividing it in four nucleosomal subregions were observed. The results obtained with FISH indicate that this monomer usually appears distributed in the terminal regions of most chromosomes and is associated to heterochromatin blocks observed after DAPI staining. These observations are discussed in relation to the satellite DNA evolution and compared with other features observed in several plant groups.

Diversity of Aspergillus oryzae genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.

Pembuatan Perpustakaan Genom Kedelai (Glycine max (L.) Merrill) Kultivar Lumut di dalam Fag Lambda

Genomic library of soybean cv. Lumut had been constructed in l phage vector. Partial digestion of total DNA by 0.014 units Sau3AI per mg DNA at 37 o C for 30 minutes gave long fragment DNA. Ligation between insert DNA and l vector with molarity ratio of insert DNA to l vector = 2.9 : 1 followed by phage packaging, resulted in the titer of 2.8x10 4 to 3.6x10 5 pfu for 0.5 mg insert plant DNA. Among them, 60 to 91% were recombinants depending on packaging extract protein. By using 1 mg insert plant DNA and packaging extract protein from Stratagene, we obtained 6.6x10 5 pfu recombinant. Since the length of insert DNAs in l recombinant phages were 15 kb in average and the genome size of diploid soybean was 2.23x10 6 kb and 90% of the genom was present in 2 copies, then the genomic library constructed had contained all genome of soybean cv. Lumut.

Diversity of <i>Aspergillus oryzae</i> genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.

Restriction fragment length polymorphism of mitochondrial genome of the entomopathogenic fungus Beauveria bassiana reveals high intraspecific variation

Beauveria bassiana is an entomopathogenic fungus with a growing potential for pest control in different agro-ecosystems worldwide. Such potential brings the necessity of developing a strain specific typing system. In a previous study, we reported the identification of molecular variants in mitochondrial DNA (mtDNA) polymorphism in 15 North American isolates. Results indicated a highly conserved mitochondrial genome showing only two mitochondrial genotypes (mitotypes). In this study we used whole genomic DNA from 18 isolates of B. bassiana, two unidentified Beauveria spp., and one each of B. amorpha, B. cylindrospora and B. nivea from more diverse origins. By doing single- and double-restriction enzyme digestion of total genomic DNA with EcoRI, and HindIII and then probing with BbmtE2, the predominance of mitotypes A and B was observed again, along with three newly described mitotypes (C to E). Additionally, by using whole B. bassiana mtDNA digested with HpaII as probe, we further demonstrate up to nine different mitotypes within B. bassiana. With either of the two probes, distinguished between members of the genus Beauveria and from Paecilomyces farinosus and Metarhizium anisopliae. Phylogenetic analysis could not however distinguish B. amorpha and B. nivea isolates from B. bassiana, suggesting a close genetic relation between the three species of the genus. Altogether, these results show high variability in mitochondrial genome, which can be useful as a reliable tool for the biopesticide industry for both species and isolate specific identification.

IDENTIFYING HOST STRAINS OF FALL ARMYWORM (LEPIDOPTERA\ NOCTUIDAE) IN FLORIDA USING MITOCHONDRIAL MARKERS

Two molecular techniques were used to identify host strains of fall armyworm, Spodoptera frugiperda (J. E. Smith) from male moths captured in pheromone-baited traps in north-central and central Florida. Moths collected in 1998 were analyzed using direct detection of mitochondrial DNA (mtDNA) RFLPs generated from restriction endonuclease digestion of total DNA, while moths collected in 2000 and 2001 were analyzed using a mitochondrial cytochrome oxidase subunit I (COI) gene PCR-RFLP marker. Both techniques could distinguish between rice and corn strain moths, however, the COI PCR-RFLP marker was more robust as indicated by a time interval experiment that showed that moths held for up to 15 days in a “bucket trap” could still be used for strain diagnosis. In a field study, our strategy gave results consistent with expectations. Rice strain moths were common in habitats with large areas of small grasses, corn strain moths were common in large areas planted to corn, and habitats with mixed large- and small-grass plantings contained both strains. Our methodology of combining pheromone traps with PCR-RFLP analysis will provide a valuable sampling system to determine the population ecology habits and strain isolating mechanisms of fall armyworm populations in numerous habitats, including overwintering areas of southern Florida.

Aspergillus bombycis genotypes (RFLP) from silkworm cultivation

Eighteen isolates of Aspergillus bombycis from samples of dust, insect frass, and soil collected from eight silkworm rearing facilities in Japan, as well as single silkworm rearing facilities in Indonesia and Malaysia, were subjected to DNA fingerprinting. PstI digests of total genomic DNA from each isolate were probed using the pAF 28 repetitive sequence. Among 18 isolates analyzed, 7 distinct DNA fingerprint groups were identified, including GTAb-2 isolated from rearing facilities in four prefectures of Japan. Aspergillus bombycis isolates share several features in common with domesticated yellow-green aspergilli, the koji molds used in traditional Oriental food fermentations, including a loose and deep colony texture, smoothwalled stipes, and the absence of sclerotia. Although aflatoxin is unknown from koji molds, all isolates of A. bombycis produced B and G aflatoxins. Aflatoxin has been linked to increased virulence in Aspergillus disease of silkworms, and there should be strong selection for aflatoxin production among clonal populations of A. bombycis adapted to silkworm cultivation. A hypothesis is offered that A. bombycis isolates from silkworm cultivation represent highly adapted forms of yet to be discovered “wild” populations that may infect Bombyx mandarina.

Interspecific Mitochondrial DNA Restriction Fragment Length Polymorphisms in Aspergillus Section Flavi

Mitochondrial DNA restriction fragment length polymorphisms (RFLPs) are described for 64 isolates, representing 11 species of Aspergillus section Flavi. Mitochondrial DNA haplotypes were identified following digestion of total cellular DNA with the restriction enzymes HaeIII, AseI, or DraI. In general, isolates of the same species possessed identical mitochondrial DNA haplotypes. Three haplotypes were found in multiple, closely related species: one in A. flavus, A. oryzae, and A. subolivaceus; a second in A. parasiticus and A. sojae; and a third in A. tamarii and A. flavofurcatis. Four distinct haplotypes were each associated with a single species: A. nomius, A. avenaceus, A. leporis, and A. zonatus. Mitochondrial DNA haplotypes complement traditional morphological and growth criteria in making taxonomic decisions within Aspergillus section Flavi.

Interspecific mitochondrial DNA restriction fragment length polymorphisms in Aspergillus section Flavi

Mitochondrial DNA restriction fragment length polymorphisms (RFLPs) are described for 64 isolates, representing 11 species of Aspergillus section Flavi. Mitochondrial DNA haplotypes were identified following digestion of total cellular DNA with the restriction enzymes HaeIII, AseI, or DraI. In general, isolates of the same species possessed identical mitochondrial DNA haplotypes. Three haplotypes were found in multiple, closely related species: one in A. flavus, A. oryzae, and A. subolivaceus; a second in A. parasiticus and A. sojae; and a third in A. tamarii and A. flavofurcatis. Four distinct haplotypes were each associated with a single species: A. nomius, A. avenaceus, A. leporis, and A. zonatus. Mitochondrial DNA haplotypes complement traditional morphological and growth criteria in making taxonomic decisions within Aspergillus section Flavi.

Common genotypes (RFLP) within a diverse collection of yellow-green aspergilli used to produce traditional Oriental fermented foods

DNA fingerprinting was performed on 72 strains of Aspergillus oryzae and 9 strains of Aspergillus sojae isolated from chu (China) or koji (Japan) mold inoculum used in the production of traditional Oriental fermented beverages or foods including soy sauce, miso, and sake. The cultures were deposited with the ARS Culture Collection (NRRL) between 1909 and 2001. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. All strains of A. sojae that we examined produced an identical DNA fingerprint and belong to the same DNA fingerprint group (GTAo-9). Strains of A. oryzae were distributed among 41 DNA fingerprint groups, including GTAo-12 represented by 11 strains, GTAo-19 represented by 5 strains, GTAo-5 and GTAo-15 each represented by 4 strains, and GTAo-8, GTAo-17, and GTAo-24 each represented by 3 strains. Thirty-three single strain isolates of A. oryzae produced unique fingerprints. Our data offer evidence suggesting that (1) the successful domestication of A. parasiticus genotypes yielding A. sojae occurred far less frequently than among genotypes of A. flavus var. oryzae; and (2) some Aspergillus genotypes employed in different fermentations and regions were derived from a common ancestral clonal population.

Increased expression of Borrelia burgdorferi vlsE in response to human endothelial cell membranes.

RNA isolated from virulent Borrelia burgdorferi cells incubated with human endothelial or neurological tissue cells was subjected to subtractive hybridization using RNA from the same strain incubated in tissue culture medium alone. This RNA subtractive technique generated specific probes that hybridized to two restriction fragments (8.2 kb and 10 kb respectively) generated by EcoRI digestion of total plasmid DNA. The 10 kb EcoRI fragment localized to lp28-1 and was subsequently identified as the variable membrane protein-like sequence (vls) region, which includes an expression locus (vlsE) and 15 silent cassettes. vlsE encodes a 36 kDa outer surface protein that undergoes antigenic variation during animal infections. Primer extension analysis identified the 5' end of a transcript and a putative promoter for vlsE. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) suggested that the expression of vlsE increased when virulent B. burgdorferi cells were incubated with human tissue cells or purified cell membranes isolated from those same cell lines. A 138 bp region upstream of the vlsE region that was not reported in the genome sequence was sequenced using specific 32P end-labelled primers in a DNA cycle sequencing system at high annealing temperatures. Analysis revealed that it contained a 51 bp inverted repeat, which could form an extremely stable cruciform structure. Southern blots probed with the vlsE promoter/operator region indicated that part or all of this sequence could be found on other B. burgdorferi plasmids.

DNA fingerprints of Caulerpa taxifolia provide evidence for the introduction of an aquarium strain into the Mediterranean Sea and its close relationship to an Australian population

The occurrence of Caulerpa taxifolia in the Mediterranean Sea was reported for the first time in 1984. Since then the alga has spread rapidly and is now considered to be a potential threat to sublittoral ecosystems. Two hypotheses on the origin of the Mediterranean strain of C. taxifolia have been discussed in the literature. One hypothesis assumed migration of the alga from the Red Sea, the other introduction via a public aquarium. The hypothesis of a descent from an aquarium strain has been supported strongly by recent studies based on DNA sequences. The DNA fingerprints of C. taxifolia presented here also provide evidence for the descent of the Mediterranean C. taxifolia from an aquarium strain. Furthermore, the present study shows that a strain of C. taxifolia from Manly Harbour/Moreton Bay (Australia) is closely related to the aquarium/Mediterranean strain. The feasibility of detecting similar genotypes by restriction digests of total DNA is demonstrated, which will facilitate the ongoing search for further relatives of Mediterranean C. taxifolia.

An epidemiological study of Plesiomonas shigelloides diarrhoea among Japanese travellers.

Plesiomonas shigelloides is often regarded as a non-pathogenic bacterial species that is occasionally isolated from patients with diarrhoea. However, a review of travellers returning to Japan with diarrhoeal illness through Kansai Airport revealed that the incidence of P. shigelloides from microbiologically confirmed cases increased from 23.2% in 1987 to 77.8% in 1999. We carried out a descriptive epidemiological study to identify patterns associated with diarrhoea due to this organism. Selected P. shigelloides isolates from this patient group were compared by pulsed-field gel electrophoresis of SpeI total chromosomal DNA digests to determine their genetic heterogeneity. Over the study period (whole of 1996 and first 2 months of 1999), 1149 of 1659 (69.3%) patients with microbiologically confirmed gastroenteritis yielded P. shigelloides. Infection was characterized by watery diarrhoea five times per day that persisted for 3 days. No statistically significant association was found between factors such as age, gender, destination, length of trip, but multivariable logistic regression analysis revealed an association between additional symptoms (vomiting, fever, abdominal pain) age and gender. The molecular fingerprints of a selection of 39 isolates and 3 reference strains of P. shigelloides were highly variable and each had a unique profile. We conclude that although P. shigelloides infections are usually mild and self-limiting, this organism may contribute to a significant proportion of travellers' diarrhoea in the Orient. The species is characterized by great heterogeneity at the DNA level.

Genetic exchange in Metarhizium anisopliae strains coinfecting Phaedon cochleariae is revealed by molecular markers

Recombination between two strains of Metarhizium anisopliae, subcultured on artificial medium and passed through an insect host was investigated. When grown on agar culture, or passed through a host, strains did not show detectable genetic changes. When passed through an insect in the presence of the other strain, genotypic changes in the isolates, as detected by esterase electrophoresis, restriction digestion of total genomic DNA and RAPD-PCR, were apparent. The instability of molecular markers suggests recombination between strains.

Interspecific transfer of Streptomyces giant linear plasmids in sterile amended soil microcosms.

The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30 degrees C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 x 10(-4) and 3.6 x 10(-5) CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment.

Taxonomic characterization of denitrifying bacteria that degrade aromatic compounds and description of Azoarcus toluvorans sp. nov. and Azoarcus toluclasticus sp. nov.

A taxonomic characterization of twenty-one strains capable of degrading aromatic compounds under denitrifying conditions, isolated from ten different geographical locations, was performed on the basis of general morphological and physiological characteristics, cellular fatty acids, DNA base composition, small ribosomal (16S) subunit DNA sequences, whole-cell protein patterns and genomic DNA fragmentation analysis, in addition to DNA similarity estimations using hybridization methods. The collection of strains was subdivided into a number of different groups. A first group, consisting of four strains, could be assigned to the previously described species Azoarcus tolulyticus. A second group (five strains) had DNA which reannealed highly to that of strains of the first group, and it is considered to represent a genomovar of A. tolulyticus. The third and fourth groups, composed of a total of five strains, represent a new species of Azoarcus, Azoarcus toluclasticus (group 3) and a genomovar of this species (group 4), respectively. Finally, the fifth group, with two strains, corresponds to another new species of the genus Azoarcus, Azoarcus toluvorans. In addition to these five groups, the collection includes five individual strains perhaps representing as many different new species. The above classification is partially consistent with the results of approaches other than DNA-DNA hybridization (electrophoretic patterns of whole-cell proteins and of the fragments obtained after digestion of total DNA with infrequently cutting restriction enzymes). On the other hand, no correlation of these groupings was found in terms of the cellular fatty acid composition. It is also unfortunate that no simple sets of easily determinable phenotypic properties could be defined as being characteristic of each of the groups.