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91.
Wilfried Bautsch 《Molecular biotechnology》1994,2(1):29-44
Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome.
The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome
map may readily be solved by applying twodimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful
methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be
applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step
protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D
gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation
of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions.
Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods. 相似文献
92.
Horizontal starch gel electrophoresis was employed to analyse genetic variation, at 19 isozyme loci, in six putatively related species of gobies ( Brachygobius aggregatus, B. doriae, Pandaka lidwilli, Pseudogobius olorum, Pseudogobius sp. and Redigobius sp.), using seven other gobiine species as an outgroup. Allozyme data was analysed both cladistically and phenetically, all treatments confirming the monophyly of the group. Pandaka is confirmed as the sister genus to Brachygobius , these genera forming a crown group, with Redigobius and Pseudogobius forming successive sister groups. This scenario is in agreement with morphological hypotheses of phylogeny. However, this lineage appears to be relatively unrelated to other groups within the Gobiinae 相似文献
93.
The populations of Gelidium canariensis (Grunow) Seoane-Camba from the Canary Islands were analyzed for genetic variability by isozyme electrophoresis in 1989 and 1990. Each population was divided into sporophytic and gametophytic subpopulations. Twenty-three to 27 putative alleles corresponding to 22 gene loci were analyzed. Sev-enteen loci were monomorphic in all six subpopulations, and five were polymorphic in at least one subpopulation. Significant deviations from Hardy-Weinberg equilibrium were found. The amount of genetic variability (percentage of polymorphic loci, mean number of alleles per locus, and average gene diversity) of haploid subpopulations was lower than that of diploid subpopulations. No correlation between genetic distance and geographical distance was found. Low genetic differentiation between sporophytic and gametophytic subpopulations of the same locality was obsewed in two populations. The low genetic diversity and genetic differentiation suggest that the genetic structure of the populations of G. canariensis from the Canary Islands is due to a combination of founder effects and the predominance of asexual reproduction. Initial differences in gene frequencies may have persisted because of insufficient time to reach a higher level of differentiation. 相似文献
94.
DAVID GOULSON 《Biological journal of the Linnean Society. Linnean Society of London》1993,49(2):127-139
Most temperate butterflies exhibit a tightly synchronized unimodal adult emergence to facilitate mate location. Exceptions are presumably subject to unusual selection pressure. This study examines the pattern of emergence in Maniola jurtina , which was found to exhibit both unimodal and bimodal emergence patterns at different sites in south-east England. The bimodal pattern was found on chalk grassland; elsewhere the emergence was unimodal. Adults from each emergence peak rarely meet, suggesting that there may be some degree of reproductive isolation. Morphological measurements and electrophoretic analysis of allozyme frequencies are carried out to quantify differentiation between emergence peaks. Captive stock was reared to examine differences in the immature stages. Butterflies from each emergence differ significantly in most morphological variables measured, those from the second peak tending to be smaller. The immature stages differ in morphology and longevity of the egg stage. Allozyme frequencies did not differ between peaks, suggesting that they are not reproductively isolated. Explanations for the maintenance of differences between emergence peaks despite gene flow are discussed. I propose that division of offspring between two emergence times may have evolved to avoid the risk inherent in placing all offspring in one peak which may be rendered inviable by temporal fluctuations in habitat quality. 相似文献
95.
Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato. Twelve wild-type and laboratory strains, representing the less agressive species O. ulmi and both of the biotypes of the more aggressive species O. novo-ulmi were studied and their karyotypes determined. Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception. Strain CESSI6K (O. novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb. This unique band was the smallest O. ulmi s. l. chromosomal DNA observed. Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype. There was no correlation between chromosome profile and species, as some O. novo-ulmi and O. ulmi strains shared common electrophoretic karyotypes. 相似文献
96.
97.
The demonstration of prophenoloxidase after electrophoresis is based on its activation by sodium dodecyl sulfate (SDS) or sodium oleate and staining the activated phenoloxidase with dopamine and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH). A rapid method is presented for demonstrating the presence of activated phenoloxidase using polyacrylamide gel electrophoresis followed by staining in the presence of SDS or sodium oleate. 相似文献
98.
Yukie Takahashi Masako Sohnaka Minoru Nakao Kenji Miyamoto Masahito Fukunaga 《Microbiology and immunology》1993,37(9):721-727
Thirty-two Borrelia isolates were obtained from the adult stage of ixodid ticks, Ixodes ovatus, collected in various localities in Japan. Borrelial isolates were cultivated and analyzed by polyacrylamide gel electrophoresis, with monoclonal antibodies, by pulsed field gel electrophoresis, and by genomic Southern hybridization. All borrelial isolates showed similar protein profiles and monoclonal antibody reactivities, while plasmid profiles were rather diverse. Genomic hybridization using rRNA gene probes demonstrated the genetic similarities of those isolates. We found no significant differences among the borrelial isolates tested, and the restriction fragment length polymorphism patterns of I. ovatus isolates were quite distinct from those of borrelial strains associated with Lyme disease. Therefore, the isolates of Borrelia obtained from I. ovatus were thought to fall into different genospecies. 相似文献
99.
To be able to understand cellular mechanisms, we require fully integrated data sets combining information about gene expression, protein expression, post-translational modification states, sub-cellular location and complex formation. Proteomics is a very powerful technique that can be applied to interrogate changes at the protein level. Studying this effectively requires specialised facilities within research institutes. Here, we describe the setting up and operation of such a facility, providing a resource for the Arabidopsis and Drosophila research communities. 相似文献
100.
The population genetic structure of the butterfly Melitaea didyma was studied along the northern distribution range border in Central Germany by means of allozyme electrophoresis. Individuals were sampled from a total of 21 habitat patches from four regions, and two provinces. Sampling was designed to estimate local vs. regional differentiation. High levels of variability were found, H e = 0.14–0.21. The mean expected sample heterozygosity from one region, Mosel, was significantly lower than from the Hammelburg region, H e = 0.17 and 0.19, respectively. Two hierarchical levels of genetic differentiation were found. Within regions individuals sampled from different patches behaved as belonging to one population with high levels of gene flow (Hammelburg F ST = 0.015, Mosel F ST = 0.044), though local isolation barriers did create a substructuring of these populations. The inbreeding coefficients, F IS , were constant over all sample levels, suggesting a similar distribution of habitat patches within regions. Between regions gene flow was limited. An isolation by distance analysis indicated that the hierarchical structure, at the provincial level, may be breaking down due to isolation of regional populations. A more general observation was that the sampling design may greatly have influenced the estimation of genetic differentiation. Depending on which samples were included, overall F ST estimates ranged from 0.059–0.090. 相似文献