bluuebunny/crossref_metadata_2025_split huggingface dataset:
Outgroup heteroduplex analysis using temperature gradient gel electrophoresis: high resolution, large scale, screening of DNA variation in the mitochondrial control region
Authors: CAMPBELL N. J. H. , HARRISS F. C. , ELPHINSTONE M. S. , BAVERSTOCK P. R. 
Published: April 2008

AbstractThe ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol ‐ Outgroup Heteroduplex Analysis (OHA). We assess this technique's sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433‐base‐pair fragment of Control Region mitochondrial DNA from Melomys cerbinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.
Mitochondrial DNA variation, population structure, and evolution of mountain sheep in the south‐western United States and Mexico
Authors: II R. R. RAMEY 
Published: April 2008

AbstractIn the south‐western United States and Mexico, mountain sheep were found to have low levels of mtDNA sequence divergence among haplotypes and low overall nucleotide diversity. Potential causes for this low diversity are discussed. Significant differences in mtDNA haplotype distributions over short distances and high values of NST on a local scale appear to be a result of the tendency of female mountain sheep to disperse less frequently and over shorter distances than males. A lack of concordance between mtDNA haplotype distributions and assumed subspecies boundaries suggest that some taxonomic labels need to be revised.
Molecular structure of the Frankia spp. nifD—K intergenic spacer and design of Frankia genus compatible primer
Authors: NALIN R. , DOMENACH A. M. , NORMAND P. 
Published: April 2008

AbstractThe nifD—K intergenic spacer (IGS) of ArI3 and ACoN24d were found to have a length 265 and 199 nucleotides, respectively. They are markedly less conserved than the two neighbouring genes and have, in some instances, a repeated structure reminiscent of an insertion event The repeated sequence and the IGSs have no detectable homology with sequences in DNA databanks. The IGS has a stem‐loop structure with a low folding energy, lower than that between nifH and nifD. No convincing alignment of IGS sequences could be obtained among Frankia strains. Only between ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structure. No promoter could be detected in the IGSs. The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleotides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN7). The codon usage was similar to that of previously sequenced Frankia genes with a strong bias toward G‐ and C‐ending codons except in the case of glycine where GGT is frequent. Alignment of the three Frankia nifK sequences (EUN1f; ArI3 and ACoN24d) with those of other nitrogen‐fixing bacteria permitted detection of a sequence conserved among the three Frankia strains but absent in the other sequences. A primer targeted to that region in combination with FGPD807‐85 amplified the nifD—K IGS sequences of all Frankia strains (except the non‐nitrogen‐fixing Frankia strains CN3 and AgB1‐9) and yet failed to amplify DNA of all other nitrogen–fixing bacteria. Conversely, the failure of primer FGPK700′‐92 to amplify Alnus‐infective strains could be explained by point mutations in the 3′ part of the primer.
Use of the PCR and fluorescent probes to recover SSU rRNA gene sequences from single cells of the ciliate protozoon Spathidiutn
Authors: DYAL P. L. , HOPE S. , ROBERTS D. M. , EMBLEY T. M. 
Published: April 2008

AbstractA two‐stage heminested PCR approach was developed to amplify small subunit (SSU) rDNA sequences, via two overlapping fragments, from single cells of microbial eucary‐otes. The method was evaluated using the ciliate protozoon Spathidiutn when PCR products were obtained from nine of 10 cells tested. Southern blotting demonstrated that all fragments contained the same sequence in a region of SSU rDNA which is normally highly variable between species. A fluorescent oligonucleotide probe was used to demonstrate that this sequence also occurred in fixed cells of Spathidiutn. Fixatives containing mercuric salts preserved cell shape and allowed probe binding with little background auto‐fluorescence. The Spathidiutn sequence is closely related to that from the haptorid Homalozoon vermiculare.
Nucleic‐acid‐based methods for monitoring the performance of in situ bioremediation
Authors: BROCKMAN F. J. 
Published: April 2008

AbstractOver the last decade, major advancements have occurred in the application of nucleic‐acid‐based methods to detect and determine the levels of catabolic genes in environmental samples. Studies have focused on validating methods in microcosms, studying changes in the structure and expression of microbial communities in response to contaminants, and improving the sensitivity of the methods. Only in the last few years have these methods transitioned from development and validation to efforts to apply these methods for monitoring in situ bioremediation. Methods that analyse nucleic acids extracted from environmental samples are of value to bioremediation because they allow analysis independent of the artefacts that can arise from laboratory biodegradative potential assays and laboratory culture‐based enumerations and from the inability to culture a large proportion of the micro‐organisms in the environment In theory, these methods enable a more comprehensive perspective, and a more defensible interpretation, of the microbial community response to intrinsic and engineered bioremediation processes. Results from the first studies applying nucleic‐acid‐based methods to intrinsic or engineered bioremediation indicate that these methods have both potential and limitations. The rapidly increasing number of cloned and sequenced catabolic genes, methodological advancements such as the ability to track specific micro‐organisms without prior sequence data, and the potential use of bioaugmentation in the field suggest that the utility of these methods for in situ bioremediation will increase in the coming years.
Extraction and purification of microbial DNA from petroleum‐contaminated soils and detection of low numbers of toluene, octane and pesticide degraders by multiplex polymerase chain reaction and Southern analysis
Authors: KNAEBEL D. B. , CRAWFORD R. L. 
Published: April 2008

AbstractWe investigated the use of multiplex polymerase chain reaction (FCR) techniques coupled with Southern analysis to detect xenobiotic‐degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pseudomonas putida mt‐2 (pWWO), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), alkB (546 bp) and tfdA (710 bp), which are found on pWWO, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide‐stained gels of the initial PCR reaction indicated detectable amplification of between 10* to 10* cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 μL) of the first reaction increased the ethidium bromide‐based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradarJve cells, at a lower detection limit of = > 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells.
Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria
Authors: BRUCE K. D. , OSBORN A. M. , PEARSON A. J. , STRIKE P. , RITCHIE D. A. 
Published: April 2008

AbstractIndividual merRTΔP regions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conserved mer sequences of Tn501, Tn21 and pMER419. Soil and sediment samples were taken from four sites in the British Isles; one ‘pristine’ (SB) and three polluted (SO, SE, T2) with respect to mercury. The sizes of the PCR products amplified (= 1 kb) were consistent with their generation from mer determinants related to the archetypal elements found in Gram negative bacteria. Forty‐five individual clones of sequences obtained from these four sites were isolated which hybridized (> 70% homology) to a merRTΔP probe from Tn501. The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling. Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites. UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified. The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation. Variation of mer genes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic variation.
Gene transfer from a bacterium injected into an aquifer to an indigenous bacterium
Authors: ZHOU J. Z. , TIEDJE J. M. 
Published: April 2008

AbstractTwo novel 3‐chlorobenzoate‐degrading bacteria were previously isolated from an aquifer in which no such bacteria could be enriched prior to the introduction of the 3‐chlorobenzoate‐degrading strain, Pseudomonas sp. B13. To understand the origin of 3‐chlorobenzoate‐degrading genes in the two novel isolates, the 16S ribosomal RNA, clcD (dienelactone hydrolase) and clcA (chlorocatechol oxygenase) genes from these bacteria were amplified and sequenced. The partial 16S rRNA gene sequences and REP‐PCR patterns showed that these two novel isolates were identical but differed from strain B13. Phylogenetic analyses revealed that the novel isolates were closely related to Alcaligenes eutrophus in the beta subclass of the Proteobacteria, whereas strain B13 was related to Pseudomonas aeruginosa and P. mendocina in the gamma subclass of the Proteobacteria. In contrast, the clcD and clcA gene sequences were identical on strain B13 and these two isolates, indicating that the 3‐chlorobenzoate‐degrading genes were transferred from strain B13 to these isolates. What cannot be established is when this transfer occurred.
Microsatellite variation in grey seals (Halichoerus grypus) shows evidence of genetic differentiation between two British breeding colonies
Authors: ALLEN P. J. , AMOS W. , POMEROY P. P. , TWISS S. D. 
Published: April 2008

AbstractEight highly variable microsatellite loci were used to examine the genetic variability and differentiation of grey seals (Halichoerus grypus) at two widely spaced British breeding colonies. Samples were collected from adults and pups on the island of North Rona, off the north‐west coast of Scotland, and on the Isle of May, situated at the mouth of the Firth of Forth on the east coast Highly significant differences in allele frequencies between these two sites were found for all eight loci, indicating considerable genetic differentiation. Thus, although grey seals are known to range over very large areas outside the breeding season, site fidelity of adults and philopatry of pups for these breeding colonies must be sufficiently common to have effects, through genetic drift, at the sub‐population level. Migration rate was estimated using Wrighf's fixation index (FST), Slatkin's private alleles model and the new statistic, RST, which is analogous to (FST) but which takes into account the process of microsatellite mutation. An almost 8‐fold discrepancy between the values we obtained provides cautionary evidence that microsatellite loci may contravene one or more of the assumptions on which these methods are based.
A PCR test for avian malaria in Hawaiian birds
Authors: FELDMAN R. A. , FREED L. A. , CANN R. L. 
Published: April 2008

AbstractThe decline of native Hawaiian forest birds since European contact is attributed to factors ranging from habitat destruction to interactions with introduced species. Remaining populations of Hawaiian honeycreepers (Fringillidae: Drepanidinae) are most abundant and diverse in high elevation refuges above the normal range of disease‐carrying mosquitoes. Challenge experiments suggest that honeycreepers are highly susceptible to avian malaria (Plasmodium sp.) but resistance exists in some species. In order to detect low levels of malarial infection and quantify prevalence of Plasmodium in high elevation natural populations of Hawaiian birds, a polymerase chain reaction (PCR) based diagnostic test was developed that identifies rRNA genes of Plasmodium in avian blood samples. Quantitative competitive PCR (QC‐PCR) experiments indicate that the detection limit of our test is an order of magnitude greater than that reported for human malaria DNA blot tests. Compared with standard histological methods, the PCR test detected a higher prevalence of diseased birds at mid‐elevations. Malaria was detected in three species of native birds living in a high elevation wildlife refuge on the island of Hawaii and in four species from Maui. Our results show that avian malaria is more widespread in Hawaiian forests than previously thought, a finding that has important conservation implications for these threatened species.
Genetic differentiation in Fomitopsis pinicola (Schwarts: Fr.) Karst studied by means of arbitrary primed‐PCR
Authors: HÖGBERG N. , STENLID J. , KARLSSON J. O. 
Published: April 2008

AbstractGeneric variation within and among one Finnish and three Swedish populations of Fomitopsis pinicola (Schwarts: Fr.) Karst. were studied by amplifying DNA from hap‐loid isolates originating from single spore cultures using two arbitrary primers. Analysis offspring from single fruit bodies revealed only three pairs of codominant alleles among 42 variable genetic markers, the remaining 38 segregated independently. Genetic similarity was measured in terms of Euclidean distance. Individuals in the Finnish population tended to form a distinct cluster in the principal component analysis. Variation within and among populations/regions was partitioned by Analysis of Molecular Variance — AMOVA. Within population variation accounted for 91.6% of the total genetic variation. The remaining 7.68% was accounted for by variation between the Finnish population and each of the three Swedish ones. Variation among the Swedish populations accounted for only 0.72% of the total variation. Wright's Fst was 0.17 for all four populations and 0.13 for the three Swedish populations. These relatively low values indicate that there is gene flow among all populations or that they are derived from a common ancestral population. The observed pattern of genetic variation is probably the result of effective spore dispersal and the continuous distribution of this common early successional species.
The effects on genetic variability following a recent colonization event: the Australian sheep blowfly, Lucilia cuprina arrives in New Zealand
Authors: GLEESON D. M. 
Published: April 2008

AbstractThe Australian sheep blowfly, Lucilia cuprina, was first identified in New Zealand in 1988 and is now found to have spread throughout many sheep‐farming regions. L. cuprina is estimated to have been present in New Zealand < 20 years, while in Australia L. cuprina has been estimated present > 100 years. The aim of this study was to determine the genetic effects of colonization of L. cuprina and to compare populations of L. cuprina from these two countries in terms of genetic variability and differentiation. Allozyme electrophoresis was used which revealed variability at six loci. 1680 blowflies were examined from 56 sites throughout L. cuprina's range in both countries. Genetic variability at each locus in terms of allele composition was found to be high and genetic differentiation varied considerably in New Zealand in comparison to Australia. Temporal sampling in New Zealand suggests seasonal fluctuations of population size in the recently colonized region of the South Island.
Intraspecific phylogeography of the gopher tortoise, Gopherus polyphemus: RFLP analysis of amplified mtDNA segments
Authors: OSENTOSKI M. F. , LAMB T. 
Published: April 2008

AbstractThe slow rate of mtDNA evolution in turtles poses a limitation on the levels of intraspecific variation detectable by conventional restriction fragment surveys. We examined mtDNA variation in the gopher tortoise (Gopherus polyphemus) using an alternative restriction assay, one in which PCR‐amplified segments of the mitochondrial genome were digested with tetranucleotide‐site endonucleases. Restriction fragment polymorphisms representing four amplified regions were analysed to evaluate population genetic structure among 112 tortoises throughout the species' range. Thirty‐six haplotypes were identified, and three major geographical assemblages (Eastern, Western, and Mid‐Florida) were resolved by UPGMA and parsimony analyses. Eastern and Western assemblages abut near the Apalachicola drainage, whereas the Mid‐Florida assemblage appears restricted to the Brooksville Ridge. The Eastern/Western assemblage boundary is remarkably congruent with phylogeographic profiles for eight additional species from the southeastern U.S., representing both freshwater and terrestrial realms.
Using chloroplast DNA to trace postglacial migration routes of oaks into Britain
Authors: FERRIS C. , OLIVER R. P. , DAVY A. J. , HEWITT G. M. 
Published: April 2008

AbstractPostglacial migration is a major factor responsible for the patterns of genetic variation we see in natural populations. Fossil pollen data indicate that early postglacial colonists such as oak, were able to take both western and eastern migration routes into Britain. Analysis at a finer level is now permitted by the use of modern molecular techniques. A 13‐bp duplication in the chloroplast tRNALeul intron occurs in natural populations of East Anglian oaks, but is not found in other parts of Britain or from mainland Europe. The distribution of this marker suggests that the mutation occurred either in southern England, or during migration from the mainland, and became fixed in a source population from which East Anglia was colonized. Planting of non‐native trees for roadside boundaries and in the grounds of old houses and estates, explains the absence of the marker from some East Anglian oaks.
Handicapped males and extrapair paternity in pied flycatchers: a study using microsatellite markers
Authors: ELLEGREN H. , LIFJELD J. T. , SLAGSVOLD T. , PRIMMER C. R. 
Published: April 2008

AbstractWe report an attempt to induce extrapair copulations and fertilizations in a species with a low intensity of sperm competition, the pied flycatcher Ficedula hypoleuca. Shortly after pair formation males were made less attractive to females by removing certain wing and tail feathers. Earlier research has shown that this manipulation reduces a male's pairing success. The idea was to test whether females mated to such males (N= 9) were more likely to obtain extrapair fertilizations than females mated to unmanipulated controls (N= 9). Paternity testing was carried out on all 98 young in the 18 broods, using a set of six microsatellite markers isolated from the species. Extrapair fertilizations were revealed in only three (17%) broods; two broods of handicapped males and one of a control male. A total of seven (7%) offspring were not genetically related to their putative father, a level which agrees well with results of other studies of this and other populations. We conclude that there was no evidence to suggest that the fertilization pattern was altered by the experimental manipulation. One reason for the lack of response could be that female mate choice in this species is based on male phenotypic, and not genotypic, quality.
Mitochondrial DNA variation and population genetic structure of the northern redbelly dace (Phoxinus eos)
Authors: TOLINE C. A. , BAKER A. J. 
Published: April 2008

AbstractTwo sections of the control region and the genes coding for NADH dehydrogenase sub‐units 5 and 6 (ND‐5/6) of mitochondrial DNA (mtDNA) were amplified from Phoxinus eos with the polymerase chain reaction. Both sections of the control region were sequenced directly while the ND‐5/6 fragment was sequenced in from each end only. Additionally, the entire ND‐5/6 fragment was examined for sequence variation using RFLP analysis. No sequence variation was detected in the control region among 70 individuals sampled from 18 populations across three Ontario regions (Spanish River, Madawaska R. and Cataraqui R.). To examine ND‐5/6 variation, a total of 75 individuals were sampled from five populations representing two of the three regions (Madawaska River and Cataraqui R.). Six haplotypes were detected by direct sequencing and four by RFLP analysis. Estimates of population subdivision from RFLP data, sequence analysis, and the two data sets combined for the ND‐5/6 fragment, suggest that gene flow is restricted within and between regions. However, estimates of sequence divergence for both sequence and RFLP analysis of this fragment suggested that populations were either founded by already differentiated populations or that populations were founded by a single stock and divergence between regions occurred prior to isolation of populations within regions. These estimates of population structure are much greater than those obtained from allozyme analysis. Additionally, high levels of heterozygosity in nuclear DNA, but low mtDNA diversity suggests that populations have experienced reductions in population size sufficient to reduce only mtDNA variation. Random lineage extinction and limited time for the accumulation of new mutations are likely responsible for low levels of mtDNA variation in ND‐5/6 and the control region, while functional constraints may limit variation more than expected in the control region in dace and other fishes.