PCR fingerprinting for epidemiological studies of Staphylococcus aureus

Staphylococcus aureus isolates (n = 126), collected during two different periods from patients hospitalised in pediatric wards, were analysed using polymerase chain reaction (PCR) mediated genotyping. These isolates were compared with 29 isolates from individuals attending the out-patient clinic of the same hospital and 13 isolates from pediatric hospital personnel. Within a group of 99 isolates gathered from 48 individuals during surveillance period I, 22 distinct genotypes were identified by application of two PCR assays. Among the 58 isolates collected in surveillance period II from pediatric and out-clinic patients, 25 genotypes were detected by a single PCR assay only. Based on these results it was demonstrated that patients can be colonised with multiple strains that may persist in a certain anatomical location for prolonged periods of time. It is shown that persistence of a S. aureus strain in a pediatric ward can be deduced from the PCR genotyping studies. As such PCR can be used for longitudinal monitoring of bacterial infections in hospital departments, analysis of patient-to-patient and personnel-to-patient transmission and for detection of genetic variation in general in S. aureus. Also, isolate-specific DNA probes can be generated for S. aureus by PCR genotyping. The probes can be used for the recognition of re-emerging S. aureus epidemics.     

Analysis by Polymerase Chain Reaction of α1 and α6 GABAA Receptor Subunit mRNAs in Individual Cerebellar Neurons After Whole-Cell Recordings

Abstract: With the use of the single-cell polymerase chain reaction (PCR), the GABA_A receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to α1 and α6 GABA_A receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, α1 subunit mRNA but not α6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the α1 and α6 GABA_A receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of α6 subunit mRNA.     

Rank and sequence in Caspar Bauhin's Pinax

CAIN, A. J., 1994. Rank and sequence in Caspar Bauhin's Pinax. Bauhin's consistent use of genera, species and binominals, applauded by historians as anticipating Linnaeus's theory and practice, does not appear on closer examination to be intended as anything of the sort. His use of the terms genus and species is as in Aristotelian logic, with a shifting reference, at all taxonomic levels. His typographical layout, emphasizing (but far from invariably employing) single-word names for effectively generic entities, often qualified by ‘and its species’, gives the impression of Linnaean practice, and coincides with it not infrequently, but not with Linnaean theory. The main entities for which it can be said that Bauhin uses fairly consistently a biverbal binominal name-phrase, like Linnaeus' trivial names, were in fact in Linnaeus's eyes two levels of supraspecific groupings. The main entities in Bauhin which Linnaeus recognized as species, as is shown by his quotations in the Species plantarum, are subdivisions of his biverbally or nearly biverbally named groupings, but themselves have multiverbal names. These correspond closely to Linnaeus's diagnostic specific names, not at all to his biverbal trivial names. Bauhin probably had no conception of the species and genus as ranks in the modern sense, first adumbrated by Tournefort and utilized by Linnaeus. Bauhin certainly tried to group forms by natural affinity, as did Theophrastus before him and Linnaeus afterwards. Not being alerted to the importance of the details of the flower and fruit, he used what characters he could find, notably, but not by any means exclusively, leaf shape. He composed the Pinax as a nomenclatural concordance to earlier authors, notably Dioscorides, Theophrastus and Pliny. He retained the sequence of major groups of Theophrastus (as the greatest authority on plants) but reversed it to start with the best-known plants, grasses. Where Theophrastus gave no help, in the cryptogams, Bauhin inserted as a pendant his own series from ferns down to fungi, using the Aristotelian principles of the gradation of forms. His overall arrangement, therefore, is not a simple progression but a chain with pendants. Bauhin is far closer to earlier authors than to Linnaeus, but his typography, along with other authors, may well have helped to incite Linnaeus to a more rigorous and consistent use of ranked groups and biverbal names.     

Phylogenetic relationships of the garter snakes based on DNA sequence and allozyme variation

We estimated phylogenetic relationships among 26 species of garter snakes (genus Thamnophis ) using allozyme and mitochondrial cytochrome b gene nucleotide sequence variation. Parsimony analyses of the two data sets give substantially different estimates of phylogeny. Several lines of evidence indicate that much of this conflict is due to error associated with the restricted number of characters in each data set. Such sampling error may be reduced by combining all the characters; we therefore present an estimate of phylogeny based on parsimony analysis of all the data combined. All our analyses support several conclusions in conflict with previous views: a very distant relationship between T.errans and T. elegans , non-monophyly of the elegans group (even excluding T: errans ), and nesting of the form validus (previously considered a member of the genus Nerodia ) within Thamnophis.
The combined analysis gives an almost fully resolved tree. However, bootstrapping indicates only weak support for many clades in this tree. Furthermore, paraphyly of the assemblages of cytochrome b gene lineages within T. elegans and T. radix indicate the potential for discordance between the mitochondrial DNA (mtDNA) and species phylogenies through the sorting of ancestral mtDNA polymorphisms. These problems suggest the need for assaying additional characters, especially ones likely to be independent of those used in the present study.     

Molecular identification of the ten subunits of cytochrome-c reductase from potato mitochondria

Cytochrome-c reductase (EC 1.10.2.2.) from Solanum tuberosum L. comprises ten subunits with apparent molecular sizes of 55, 53, 51, 35, 33, 25, 14, 12, 11 and 10 kDa on 14% SDS-PAGE. The identity of the subunits was analysed by direct amino-acid sequencing via cyclic Edman degradation. A large-scale purification procedure for the enzyme complex based on affinity chromatography and gelfiltraton is described. All subunits were enzymatically fragmented and the generated peptides were separated by reverse-phase HPLC. Complete or partial sequence determination of 33 peptides comprising a total of nearly 500 amino acids showed, that cytochrome-c reductase from potato contains three respiratory proteins (cytochrome b, cytochrome c ₁ and the Rieske iron-sulfur protein), four small proteins with molecular sizes below 15 kDa (so-called Q-binding, hinge, cytochrome-c ₁-linked and core-linked proteins) and three proteins in the 50-kDa range which show similarity to members of the core/PEP/MPP protein family (core/processing enhancing protein/mitochondrial processing peptidase). In fact these subunits show highest sequence identity either to MPP or PEP, which is in line with earlier findings, that isolated cytochrome-c reductase from potato exhibits processing activity towards mitochondrial precursor proteins.Abbreviations MPP mitochondrial processing peptidase - PEP processing enhancing protein This research was supported by the Deutsche Forschungsgemeinschaft.     

Divergence between the Anoas of Sulawesi and the Asiatic Water buffaloes, inferred from their complete amino acid sequences of hemoglobin ß chains

Four complete amino acid sequences of hemoglobin β chains were determined for the swamp and the river types of the Asiatic water buffalo (Bubalus bubalis) and two species of the subgenus Anoa in Bubalus; B. (A.) depressicornis (H. Smith, 1827), the lowland anoa, and B. (A.) quarlesi (Ouwens, 1910), the mountain anoa. The two types of the bubalis were identical in the 145 amino acid residues of the β chains and, compared to this sequence, the two residues were substituted in the depressicornis (β49Thr → Ser and 134Ala → Thr) and the five were in the quarlesi (β53Val → Ile, 74Met → Ile, 111Val → Ile, 115Arg → His and 134Ala → Thr). While both Anoa species diverged from the bubalis by the β134Ala → Thr, they differed from each other by the five substitutions. The Anoa species are endemic to Sulawesi of Indonesia. Their speciation and the present coexistence were discussed with reference to probable immigrations of two ancestral Anoa species to Sulawesi at so long interval that had caused a reproductive isolation between the two wild animals. The earlier immigrants were postulated to be ancestral to the quarlesi and the later ones to the depressicornis.     

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.     

Mitochondrial DNA control region polymorphisms: genetic markers for ecological studies of marine turtles

We describe a rapid and sensitive method for the detection of population-specific genetic markers in mitochondrial DNA (mtDNA) and the use of such markers to analyse population structure of marine turtles. A series of oligonucleotide primers specific for the amplification of the mtDNA control region in Cheloniid turtles were designed from preliminary sequence data. Using two of these primers, a 384–385-bp sequence was amplified from the 5′ portion of the mtDNA control region of 15 green turtles Chelonia mydas from 12 different Indo-Pacific rookeries. Fourteen of the 15 individuals, including some with identical whole-genome restriction fragment patterns, had sequences that differed by one or more base substitutions. Analysis of sequence variation among individuals identified a total of 41 nucleotide substitutions and a 1-bp insertion/deletion. Comparison with evidence from whole-genome restriction enzyme analysis of the same individuals indicated that this portion of the control region is evolving approximately eight times faster than the average rate and that the sequence analysis detected approximately one fifth of the total variation present in the genome. Restriction enzyme analysis of amplified products from an additional 256 individuals revealed significant geographic structuring in the distribution of mtDNA genotypes among five of the 10 rookeries surveyed extensively. Additional geographic structuring of genotypes was identified through denaturing gradient gel electrophoresis (DGGE) of amplified products. Only two of the 10 rookeries surveyed could not be differentiated, indicating that the Indo-Pacific C. mydas include a number of genetically differentiated populations, with minimal female-mediated gene flow among them. Important applications for genetic markers in the conservation and management of marine turtles include the identification of appropriate demographic units for research and management (i.e. genetically discrete populations) and assessment of the composition of feeding and harvested populations.     

The use of PCR to monitor the population abundance of six human intestinal bacterial species in an in vitro semicontinuous culture system

Abstract Six PCR primer sets complementary to the 16S rDNAs (rRNA genes) were developed and shown to be specific for the following anaerobic bacteria: Clostridium clostridiiforme, C. perfringens, C. leptum, Bacteroides vulgatus, B. distasonis , and B. thetaiotaomicron , respectively. These primers were used for PCR to detect and monitor the bacteria in a semicontinuous culture system designed to mimic intestinal microflora in the human gastrointestinal tract. Except for C. perfringens , the five species of Bacteroides and Clostridia present in the in vitro culture system were detected by the PCR, and the titers varied from 10⁻² to 10⁻⁶ dilutions. The role of azo dye reduction by these bacterial species in the system was examined and discussed.