Restriction Fragment Patterns Research Articles

Maternal Inheritance of Chloroplast DNA in Interspecific Crosses of Bromus

Inheritance of chloroplast DNA (cpDNA) was examined in 41 F1 progeny obtained from the following interspecific Bromus crosses: Bromus arvensis (2n = 14) × B. inermis (2n = 4x = 28); B. arvensis × B. inermis (2n = 8x = 56); B. arvensis × B. erectus (2n = 6x = 42); B. arvensis × B. erectus (2n = 8x = 56); B. arvensis × B. erectus (2n = 10x = 70). Chloroplast DNA of the parental species was digested with BamHI, EcoRI and HindIII and species-specific restriction fragment length polymorphisms were identified by observation of ethidium bromide stained agarose gels as well as by filter hybridization experiments involving heterologous cloned barley cpDNA probes. The stability of these point mutations was verified by examining the cpDNA restriction patterns of at least 28 individual plants raised from seed of each of the parental species. No intraspecific cpDNA variability was detected. All the F1 progeny examined exhibited the cpDNA restriction fragment patterns of the female parent. There was no evidence of any paternal or biparental cpDNA inheritance. The results provided evidence for the uniparental-maternal inheritance of cpDNA in the Bromus crosses examined.

Improved preparation of high molecular weight DNA for pulsed-field gel electrophoresis from mycobacteria

Molecular typing is now widely used to aid and supplement conventional epidemiological studies of mycobacterial diseases. Pulsed-field gel electrophoresis (PFGE), in which the entire genome can be represented as a distinct pattern of DNA restriction fragments, is a particularly powerful tool in epidemiology for the determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. Application of PFGE to the study of mycobacterial diseases has been limited because isolation of high-quality genomic DNA from mycobacterial sources has proved problematic. Here we report a simple, highly effective method for the preparation of high molecular weight DNA from a range of mycobacterial species. Cultures are continuously stirred and are homogeneous. This enables accurate quantification. The presence of detergent in buffers keeps the cells in suspension throughout preparation enabling efficient lysis. In addition, it is compatible with heat-inactivation of pathogenic mycobacteria and all of the preparation procedures can be carried out with a category III facility. This standardised method of preparation of DNA from mycobacteria means that PFGE should now be evaluated as a method for typing these organisms and it may be particularly important as a means of typing less well-characterised mycobacteria for which other techniques are not available.

Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora.

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern. phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. phiEa116C contained an approximately 75-kb genome. phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. phiEa116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU.

野生カモシカの下痢原性病原体保有状況と河川水汚染との関連性

Between February 1998 and January 2000, to investigate original sources of environment agents pathogenic in human beings, we isolated and detected microorganisms from 75 animal-fecal samples and 26 river-water samples from the vicinity of Mount Zao in Yamagata Prefecture, Japan. From the Japanese serow (Capricornis crispus), we isolated five serogroups of Verotoxin-producingEscherichia coli (VTEC). Although one isolate (O91: H-) belonged to a serogroup simultaneously discovered in a human carrier, pulsed-field gel electrophoresis showed it to have a different restriction fragment pattern. Thus no direct relation between VTEC O91: H-in the serow and in human beings was suggested. The indirect immuno-fluorescence method detected Giardia cysts in 13 Japanese serows and 1 dog for a rate of between 20 and 5, 494 per gram. Down the river from Mount Zao, on the other hand, we discovered from 3 to 63 Giardia/15-18 in 2 samples. We suspect that these may result from contamination of the river by human and agricultural wastes since we detected no Giardia in 15 water samples collected at 3 points in the river in the mountainous area where the serows live. Although we detected VTEC andGiardiain wild serows, we found no proof that microorganisms in the environment cause infectious diseases in human beings. Establishing a relation between pathogenic microorganisms in humans and wild animals will require further, long-range study.

Flammulina RFLP patterns identify species and show biogeographical patterns within species

Representative collections of Flammulina were examined by restriction digests of the ribosomal RNA internally transcribed spacer region (ITS1-5.8S-ITS2). Digestion with the restriction enzymes, Hae III and Bst F51 distinguished among the major Northern Hemisphere species of Flammulina. Digestion of F. velutipes collections with two additional restriction enzymes, Bgl I and Bst UI, gave variable restriction fragment patterns and revealed biogeographically identifiable patterns. Three haplotypes of F. velutipes were identified with geographical distributions chiefly in Asia, Europe and North America. Recent transfer of haplotypes among geographical areas seems plausible based on the observed haplotype distribution.

A unique and common restriction fragment pattern of the nucleotide sequences homologous to the genome of Vf33, a filamentous bacteriophage, in pandemic strains of Vibrio parahaemolyticus O3:K6 O4:K68, and O1:K untypeable

Vf33 is a filamentous bacteriophage isolated from Vibrio parahaemolyticus. We performed Southern blot hybridization analysis to examine the distribution of Vf33-related genetic elements in the pandemic strains (O3:K6 strains isolated between 1995 and 1997, O4:K68 and O1:K untypeable strains isolated between 1997 and 1999) of V. parahaemolyticus. Nucleotide sequences homologous to the Vf33 DNA were detected in all 57 test strains including pandemic and non-pandemic strains. However, the profiles of hybridization, including the restriction fragment length polymorphism, with nine Vf33-derived DNA probes exhibited by the pandemic strains were identical and were different from those by the non-pandemic strains. The results support the hypothesis that the pandemic strains are clonal, and suggest a possibility that they have acquired (a) new gene(s) via a Vf33-like filamentous phage.

Flammulina RFLP Patterns Identify Species and Show Biogeographical Patterns within Species

Representative collections of Flammulina were examined by restriction digests of the ribosomal RNA internally transcribed spacer region (ITS1-5.8S- ITS2). Digestion with the restriction enzymes, HaeIII and Bst F51 distinguished among the major North- ern Hemisphere species of Flammulina. Digestion of E velutipes collections with two additional restriction enzymes, Bgl I and Bst UI, gave variable restriction fragment patterns and revealed biogeographically identifiable patterns. Three haplotypes of E velutipes were identified with geographical distributions chief- ly in Asia, Europe and North America. Recent trans- fer of haplotypes among geographical areas seems plausible based on the observed haplotype distribu- tion.

Fingerprinting method for phylogenetic classification and identification of microorganisms based on variation in 16S rRNA gene sequences.

The paper describes a method for the classification and identification of microorganisms based on variations in 16S rRNA sequences. The 16S rRNA is one of the most conserved molecules within a cell. The nature of the variable and spacer regions has been found to be specific to a given organism. Thus, the method presented here can be very useful for the classification and identification of microorganisms for which very little information is available. To automate the method, a comprehensive computer program called FPMAP has been developed for the analysis of restriction fragment pattern data. The method involves the restriction digestion of genomic DNA, preferably using four-cutters that may recognize 6-9 sites within the 16S rDNA. The fragments are separated on a polyacrylamide gel along with a suitable marker, then transferred into a nylon membrane and hybridized with a radiolabeled 16S rDNA probe. After autoradiography, the fragment sizes are calculated, and the data are analyzed using the FPMAP software. We demonstrate that the method can be used for identification of strains of Streptomyces and mycobacteria. The software is available from our ftp site ftp:¿imtech.chd.nic.in/pub/com/fpmap/unix/.

Discrimination of Catadromous Eels of GenusAnguillaUsing Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis of the Mitochondrial 16S Ribosomal RNA Domain

Abstract To develop a species identification method for catadromous eels of the genus Anguilla, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of a portion of the16S ribosomal RNA (rRNA) domain (1.3 kilobase pairs) in the mitochondrial genome was carried out for 18 recognized species of the genus. The restriction fragment patterns showed the species-specific haplotypes by processing with six restriction enzymes. In addition, intraspecific haplotype variation in the mitochondrial 16S rRNA domain among 10 A. marmorata collected from geographically distant populations did not reveal genetic structure within the same species, suggesting that it is a preferable marker for species identification.

Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus.

A multilocus sequence typing (MLST) scheme has been developed for Staphylococcus aureus. The sequences of internal fragments of seven housekeeping genes were obtained for 155 S. aureus isolates from patients with community-acquired and hospital-acquired invasive disease in the Oxford, United Kingdom, area. Fifty-three different allelic profiles were identified, and 17 of these were represented by at least two isolates. The MLST scheme was highly discriminatory and was validated by showing that pairs of isolates with the same allelic profile produced very similar SmaI restriction fragment patterns by pulsed-field gel electrophoresis. All 22 isolates with the most prevalent allelic profile were methicillin-resistant S. aureus (MRSA) isolates and had allelic profiles identical to that of a reference strain of the epidemic MRSA clone 16 (EMRSA-16). Four MRSA isolates that were identical in allelic profile to the other major epidemic MRSA clone prevalent in British hospitals (clone EMRSA-15) were also identified. The majority of isolates (81%) were methicillin-susceptible S. aureus (MSSA) isolates, and seven MSSA clones included five or more isolates. Three of the MSSA clones included at least five isolates from patients with community-acquired invasive disease and may represent virulent clones with an increased ability to cause disease in otherwise healthy individuals. The most prevalent MSSA clone (17 isolates) was very closely related to EMRSA-16, and the success of the latter clone at causing disease in hospitals may be due to its emergence from a virulent MSSA clone that was already a major cause of invasive disease in both the community and hospital settings. MLST provides an unambiguous method for assigning MRSA and MSSA isolates to known clones or assigning them as novel clones via the Internet.

DNA phenotypes of the blackberry biological control agent, Phragmidium violaceum , in Australia

Characterisation of populations of Phragmidium violaceum in Australia is necessary to assess the efficacy of this exotic rust fungus for the biological control of various genotypes of European blackberry (Rubus fruticosus L. aggregate). P. violaceum was introduced to Australia on at least three separate occasions: the first being an unauthorised introduction of an unknown strain or strains in the early 1980s, and the second and third being authorised releases of the biological control agent, strain F15, in 1991 and 1992. Using restriction fragment length polymorphisms (RFLP) in minisatellite DNA (M13 probe), 13 DNA phenotypes of P violaceum were identified among 18 single-pustule isolates collected from various locations in mainland Australia between 1997 and 1999. The restriction-fragment patterns and DNA band-sharing indices of similarity suggest that so-called ‘illegal’ strains predominate in populations of P violaceum and that isolate F15 did not become well established in Australia.

Phylogenetic Relationship in the Genus Rosa Based on the Restriction Enzyme Analysis of the Chloroplast DNA.

To plot the phylogenetic relationship within the genus Rosa, we compared the restriction sites of chloroplast DNAs among 32 species. With the use of 14 restriction endonucleases, restriction fragment patterns of cpDNAs were obtained from 32 lines of Rosa species and compared with each other. Three hundred thirty-five restriction sites were surveyed. Thirty-three sites were phylogenetically informative. Among the chloroplast genome types, 18 primary groups are formed. The majority rule consensus tree established by the Wagner parsimony method indicated that the species in sections Synstylase and Indica, and sections Carolinae and Cinnamomeae belong to a monophyletic clade, which agrees with the morphological classification. Sections Carolinae, Cinnamomeae, and Pimpinelifoliae lie distantly from the other sections, suggesting that these sections may have differentiated in an early stage of evolution followed by the appearance of other sections. A comparison of the previous classification of Rosa species with that in this study, revealed several discrepancies. R. gigantea, R. moschata, and R. damascena, which appear to be monophyletic, were grouped into one cluster apart from section Gallicanae, to which R. damascena was formerly classified. The present molecular data indicate that the maternal ancestor of R. centifolia 'Japonica' may be R. canina. It is proposed that R. roxburghiin and R. roxburghiin hirtula are separated, based on their differences in cpDNA. Interspecific cpDNA variation is common, a great deal of variability was detected in Rosa. Excessive dependence on one particular cytoplasm may be undesirable, we suggest that the methods described in this paper be used to survey the cytoplasmic base of modern rose cultivars.

5-Methylcytosine distribution and genome organization in triticale before and after treatment with 5-azacytidine.

Triticale (2n=6x=42) is a hybrid plant including rye (R) and wheat (A and B) genomes. Using genomic in situ hybridization with rye DNA as a probe, we found the chromosomes of the R genome were not intermixed with the wheat chromosomes in 85% of nuclei. After treatment of seedlings with low doses of the drug 5-azacytidine (5-AC), leading to hypomethylation of the DNA, the chromosomes became intermixed in 60% of nuclei; the next generation showed intermediate organization. These results correlate with previous data showing that expression of R-genome rRNA genes, normally suppressed, is activated by 5-AC treatment and remains partially activated in the next generation. The distribution of 5-methylcytosine (5-mC) was studied using an antibody to 5-mC. Methylation was detected along the lengths of all chromosomes; there were some chromosome regions with enhanced and reduced methylation, but these were not located at consistent positions, nor were there differences between R and wheat genome chromosomes. After 5-AC treatment, lower levels of methylation were detected. After 5-AC treatment, in situ hybridization with rye genomic DNA sometimes showed micronuclei of rye origin and multiple translocations between wheat and rye chromosomes. Genomic DNA was analysed using methylation-sensitive restriction enzymes and, as probes, two rDNA sequences, two tandemly organised DNA sequences from rye (pSc200 and pSc250), and copia and the gypsy group retrotransposon fragments from rye and wheat. DNA extracted immediately after 5-AC treatment was cut more by methylation-sensitive restriction enzymes than DNA from untreated seedlings. Each probe gave a characteristic restriction fragment pattern, but rye- and wheat-origin probes behaved similarly, indicating that hypomethylation was induced in both genomes. In DNA samples from leaves taken 13-41 days after treatment, RFLP (Restriction Fragment Length Polymorphism) patterns were indistinguishable from controls and 5-AC treatments with all probes. Surprising differences in hybridization patterns were seen between DNA from root tips and leaves with the copia-fragment probes.

Mitochondrial DNA Mixed-Stock Analysis of American Shad: Coastal Harvests Are Dynamic and Variable

Abstract Populations of American shad Alosa sapidissima are generally declining in size along the U.S. East Coast. Coastal harvest of migrating mixed-stock assemblages has been proposed as a possible cause of the decline, but the extent of fishing pressure on any particular stock has not been evaluated. To assess origin of shad harvested in the coastal fishery, we applied genetic mixed-stock analysis by using mitochondrial DNA (mtDNA) variation. We examined American shad from the coastal mixed fisheries off both Virginia and Maryland in 1992 and off Virginia alone in 1993 (N = 250 and 270, respectively), comparing mtDNA restriction fragment patterns for the coastal individuals with an archival database containing mtDNA genotypes of 1,734 individuals from 16 North American river stocks distributed from Florida to Canada. We used two different maximum-likelihood approaches to estimate the contributing-stock composition of oceanic mixtures, one yielding conditional estimates of stock composition, given obser...

Genome analysis of several marine, magnetotactic bacterial strains by pulsed-field gel electrophoresis.

The genomic DNA of three strains of marine magnetotactic bacteria, including two facultatively anaerobic vibrios, strains MV-1 and MV-2, and the microaerophilic coccus, strain MC-1, was analyzed by pulsed-field gel electrophoresis (PFGE). Digestion of the genomic DNA of strain MV-1 by the restriction endonucleases AvrII, BamHI, HindIII, NheI, SalI, SfiI, SgfI, SgrAI, and XbaI resulted in a large number of fragments below 400 kb that were difficult to resolve by PFGE. Digestion of MV-1 DNA with NotI and RsrII resulted in no fragments. Treatment of genomic DNA of strains MV-1 and MV-2 with PacI, PmeI, and SpeI yielded a manageable number of fragments (ca. 20) that were relatively easily resolved with PFGE, while PacI and SpeI were effective for strain MC-1. There was no evidence for the presence of plasmids and linear chromosomes in any of the strains, and strains MV-1 and MV-2 appear to contain a single, circular chromosome. Genome sizes of strains MV-1, MV-2, and MC-1 were estimated to be between 3.6 and 3.9 Mb (mean +/- SD; 3.7 +/- 0.2), 3. 3 and 3.7 Mb (3.6 +/- 0.2), and 4.3 and 4.7 Mb (4.5 +/- 0.3), respectively. The restriction fragment patterns of the vibrioid strains MV-1 and MV-2 were extremely similar, suggesting that the strains are closely related.

Restriction Enzyme Analysis of PCR Amplified rDNA as a Taxonomic Tool in Yeast Identification

A method has been developed to simplify the identification of yeast strains. We used the restriction fragment patterns of PCR-amplified 18S rRNA-coding DNA with the neighbouring ITS1 region for differentiation and identification of 169 yeast strains representing 128 species associated mainly with food, wine, beer, and soft drinks. The amplicons were digested with four different four-base-cutting restriction enzymes. To construct a database of restriction fragment patterns, the gels have been scanned and analyzed using the Molecular Analyst Fingerprint 2.0 software. The use of four enzymes proved to be sufficient for strain identification.

Molecular and kinetic evidence for allelic variants of esterase Estbeta1 in the mosquito Culex quinquefasciatus.

Elevated esterase Estbeta1 was purified from larvae of newly isolated strains of the mosquito Culex quinquefasciatus from Colombia (COL) and Trinidad (TRI) with resistance to organophosphate (OP) insecticides. Insecticide interactions were compared with those of elevated Estbeta1(2) from the OP-resistant Habana strain and the non-elevated Estbeta1(3) from the susceptible PelSS strain. On the basis of insecticide binding efficiency, all elevated Estbeta1 esterases were readily distinguishable. Differences between the EcoRI restriction fragment patterns of the amplified estbeta1 gene in COL and TRI strains compared with each other, and between amplified estbeta1(1), estbeta1(2) and the non-amplified estbeta1(3), suggest differences in their nucleotide sequence. Considering their variable insecticide binding efficiencies, these genetic differences would imply that, in contrast to estalpha2 and estbeta2, amplification of estbeta1 has occurred several times independently. Generally, the elevated Estbeta1s were more reactive with insecticides than the non-elevated Estbeta1(3). This supports the hypothesis that the elevated esterase-based mechanism confers resistance through amplification of alleles coding for esterases which have a greater specificity for the insecticides they sequester than the esterases coded by their non-amplified counterparts.

Molecular evidence supports Phytophthora quercina as a distinct species

Phytophthora auercina, a new species associated with oak decline in Europe, has been assigned to Waterhouse's Group I of Phytophthora. The level of intraspecific variation and evidence of affinities to other Group I species and another, as yet unidentified, species from oak, were examined at the molecular level using four random ten-mer primers to amplify total DNA (RAPDs). Sequences and restriction fragment length polymorphisms of a 900 bp PCR product consisting of the ITS1 and ITS2 regions, and the 5.8S subunit of ribosomal DNA were also examined to estimate relatedness to a broader range of Phytophthora species. The RAPD banding patterns of ten isolates of P. quercina from eight sites in Germany, Hungary and Italy were almost identical and distinct from all the other species tested. Their ITS restriction fragment patterns were also identical, as were the ITS sequences of four selected isolates. Phylogenetic analysis of the ITS sequence data confirmed its unique position in this section of the genus which comprises P. quercina, another five Group I species, P. infestans (Group IV) and P. nicotianae (Group II). Isolates of P. quercina formed a distinct branch at the base of this clade showing no close affinities with any other species. Such data support morphological, physiological and pathological evidence that P. quercina is distinct, although it has some affinity with the other Group I species. The results support earlier reports that Waterhouse's groupings of morphospecies do not fully correspond to phylogenetic relationships indicated by molecular studies. The unidentified Phytophthora sp. 2 from oak was closely related to P. ilicis (Group IV); both were distinct on molecular criteria from all other species in the study.

Diversity of Microorganisms Isolated from Amber.

> Abstract Claims that organisms can be cultured from amber, if substantiated, would be significant contributions to our understanding of the evolution, tenacity, and potential spread of life. Three reports on the isolation of organisms from amber have been published. Cano and Borucki recently reported the isolation of Bacillus sphaericus and Lambert et al. have described a new species designated Staphylococcus succinus from 25-40 million year old Dominican amber. These characterized organisms were phylogenetically distant from extant relatives and the Staphylococcus sp. sufficiently far removed from other extant staphylococci to be considered a new species. Here we report the culture of bacteria from Dominican and previously untested 120 million year old Israeli (Lebanese lode) amber. Twenty-seven isolates from the amber matrix have been characterized by fatty-acid profiles (FAME) and/or 16S rRNA sequencing. We also performed a terminal restriction fragment pattern (TRF) analysis of the original amber before prolonged culture by consensus primer amplification of the 16S rRNA followed by restriction enzyme digestion of the amplicons. Sample TRFs were consistent with a sparse bacterial assemblage and included at least five of the isolated organisms. Finally, we microscopically mapped the internal topography of an amber slice.http://link.springer-ny.com/link/service/journals/00248/bibs/38n1p58.html

Characterization of nosocomial Serratia marcescens isolates: Comparison Of Fouriertransform Infrared Spectroscopy With Pulsed-Field Gel Electrophoresis of Genomic Dna Fragments and Multilocus Enzyme Electrophoresis

A total of 66 Serratia marcescens isolates from 46 patients was investigated by macrorestriction using XbaI followed by pulsed-field gel electrophoresis. 7 restriction fragment patterns attributable to more than one patient and 9 individual patterns were identified. The isolates were additionally characterized by multilocus enzyme electrophoresis and Fourier-transform infrared spectroscopy. The macrorestriction patterns and the multilocus enzyme electrophoresis patterns corresponded fairly well while the classifications derived from these methods were not completely congruent. The grouping achieved by Fourier-transform infrared spectroscopy on the basis of high (> 1000) and moderately high heterogeneity values (300) was consistent with the macrorestriction results. Grouping on a lower heterogeneity level did not contribute to further discrimination. In general, Fourier-transform infrared spectroscopy was less discriminatory than the two other methods, but easier to perform. Therefore, laboratories equipped with the necessary devices may use it to rapidly select bacterial isolates for macrorestriction or other well established characterization procedures.