EcoRI Patterns Research Articles

An assessment of the relationships among species of Camelidae by satellite DNA comparisons.

Tandem satellite arrays and interspersed repetitive DNA components of the New World camelids guanaco, llama, alpaca, and vicuña and the Old World bactrian camel have been identified and compared. Southern hybridizations, using camel restriction fragments as probes, indicated that satellite DNAs in all camelids examined have been conserved since the last common ancestor about 5-10 MY ago. The hybridization profiles, however, varied from totally identical (MspI-sat) to highly differentiated (PstI-sat and EcoRI-sat) between Old and New World species. Repetitive DNA patterns specific of South American camelids were identified by most of the vicuña and guanaco probes and (a) llama and guanaco have undifferentiable patterns, supporting the view that the former is a domesticated form of the latter; (b) vicuña patterns were species-specific and in agreement with its position in a separate taxonomic unit; (c) the presence in alpaca of BamHI, TaqI and EcoRI patterns that are intermediate between those of the species above, suggested that the origin of the alpaca may be found in a cross-breed between the guanaco and vicuña.

Quinupristin/dalfopristin-resistant enterococci of the satA (vatD) and satG (vatE) genotypes from different ecological origins in Germany.

The semisynthetic streptogramin combination quinupristin/dalfopristin (Synercid) is a promising alternative for treatment of infections due to multiply resistant gram-positive bacteria including vancomycin-resistant Enterococcus faecium. Resistance is mediated by acetyltransferases SatA (VatD) or SatG (VatE). Recent papers have indicated a possible link between the use of the streptogramin virginiamycin S/M as a feed additive in commercial animal husbandry and a selection of quinupristin/dalfopristin-resistant E. faecium (QDRE). We screened manure samples from two different turkey farms and from six different pig farms (using virginiamycin), samples from a sewage water treatment plant, 24 broiler carcasses, 10 pork samples, and 200 stool samples of nonhospitalized humans for QDRE. Our strain culture collection of hospital E. faecium isolates from the last 2 years was also reviewed for QDRE. All manure and sewage samples were positive for QDRE, as well as 11 from broiler carcasses (46%), 1 from pork (10%), and 28 from human stool specimens (14%). Thirty-six hospital isolates of E. faecium exhibited resistance to quinupristin/dalfopristin. In 141 QDRE of different origin satA (vatD) and satG (vatE) genes were detected (seven isolates from humans with an unknown resistance mechanism). Streptogramin resistance determinants were tansferable in filtermating experiments for 5 of 10 satA (vatD) and 9 of 22 satG (vatE) isolates. Different EcoRI patterns of satG (vatE) plasmids and corresponding hybridizations of the satG (vatE) gene indicated nonhomologous resistance plasmids in isolates of different origin. The results of this study indicate a common gene pool for streptogramin resistance in E. faecium of different ecological origin. A selection of QDRE using the streptogramin virginiamycin S/M as a feed additive and a spread of the resistance via the food chain to humans is probable.

Species identification of oral viridans streptococci by restriction fragment polymorphism analysis of rRNA genes.

Oral streptococci formerly classified as Streptococcus sanguis have been divided into six genetic groups. Methods to identify those species by genotype are needed. This study compared restriction fragment polymorphisms of rRNA genes (ribotypes) for seven S. gordonii, three S. sanguis, four S. oralis, three S. mitis, one S. crista, and seven S. parasanguis strains classified in previous DNA hybridization studies, as well as one clinical isolate. DNA was digested with HindIII, PvuII, HindIII and PvuII combined, EcoRI, BamHI, AatII, AlwNI, and DraII. DNA fragments were hybridized with a digoxigenin-labeled cDNA probe obtained by reverse transcription of Escherichia coli 16S and 23S rRNA. S. oralis, S. mitis, and S. parasanguis all showed an isolated 2,290-bp band in AatII ribotypes that was absent from S. gordonii, S. sanguis, and S. crista. The last three groups showed species-specific bands with AatII and also with PvuII. S. oralis could be distinguished from S. mitis and S. parasanguis in AlwNI and DraII ribotypes. S. mitis and S. parasanguis could not be distinguished, since they shared multiple bands in PvuII, AlwNI, and EcoRI patterns. The clinical isolate in the panel was very similar to S. sanguis by all enzymes used. Our findings suggest that ribotyping may be useful for genotypic identification of oral viridans streptococci. Initial digests of clinical isolates might be made with AatII, followed by PvuII or AlwNI. Isolates then could be identified by comparing ribotype patterns with those of reference strains. This approach could facilitate clinical studies of these newly defined species.

De novo methylation causes a tissue-specific polymorphic EcoRI pattern at the human epidermal growth factor receptor gene

A novel restriction polymorphism, probably due to tissue-specific methylation, has been identified at the human epidermal-growth-factor-receptor (EGF-R) gene. DNA isolated from smooth muscle showed altered EcoRI restriction bands when hybridized with different fragments of the EGF-R cDNA. These bands were absent in brain or leucocyte DNA samples from the same individuals. Three restriction sites, partly resistant to cleavage by EcoRI, were characterized in muscle DNA which were not clustered but instead were scattered along the gene. The flanking sequences of one of these resistant EcoRI sites were determined. This specific EcoRI site was followed by a 3'-guanosine generating a methylatable EcoRI sequence. This suggests that the failure to digest to completion these EcoRI sites was due to modification by methylation. In addition, we noted that EcoRI sites were affected at both alleles, indicating that de novo methylation changes, and not methylation events related to genomic imprinting, would cause the muscle-specific EcoRI pattern. Also abnormal restriction fragments with XbaI were observed in muscle DNA. A large number of unrelated muscle DNA samples have been analysed, and all of them displayed an identical EcoRI polymorphic pattern, suggesting that DNA modification by de novo methylation events could be functionally relevant.

<I>Chlamydia trachomatis</I>DNAの制限酵素による解析

Twelve serovar type strains (C, D, E, F, G, H, I, J, K, L1, L2 and L3) and eight isolates (D, 4; E, 2; K, 2) of C. trachomatis were examined by restriction endonuclease analysis (REA) of DNA extracted from the elementary bodies. No difference was observed in DNA patterns of three serotypes (L1, L2 and L3) of the biovar LGV when they were digested with EcoRI and analysed by electrophoresis in a 0.6% agarose gel. The genital strains of the biovar trachoma (serovars D-K) showed similar EcoRI patterns with or without detectable differences. Serovar C of the biovar trachoma differed from the biovar LGV and the genital strains. Comparative analysis of DNAs digested with EcoRI, BamHI, HindIII, SalI and NcoI revealed that C. trachomatis isolates belonging to serovars D and K, but not E, could be subdivided into different genome types. These results suggest that DNA cleavage pattern analysis is useful for epidemiological, clinical, and taxonomic studies of C. trachomatis.

Detection of genomic variation in Providencia stuartii clinical isolates by analysis of DNA restriction fragment length polymorphisms containing rRNA cistrons.

Chromosomal DNA from 26 strains of Providencia stuartii isolated mainly in hospitals in the United Kingdom and reference strains of P. stuartii, P. rustigianii, and Proteus vulgaris were digested with the restriction endonucleases EcoRI and HindIII. After electrophoresis in agarose gels, the fragments were subjected to Southern blot hybridization analysis with a biotin-labeled cDNA probe transcribed from a mixture of 16S and 23S rRNA from P. stuartii NCTC 11800T. The pattern of bands (the rDNA fingerprint), which depended on restriction fragment length polymorphisms containing rRNA genes, was used as a measure of minor genomic variation within and between species. The P. stuartii clinical isolates had similar total digest patterns, but the rDNA fingerprints revealed some heterogeneity between strains, with EcoRI digests providing better strain discrimination than HindIII. Such rDNA fingerprints comprised between five and seven bands with sizes in the range of 5 to 28 kilobases. The 11 different EcoRI patterns were compared by numerical analysis, and several groups or subgroups of strains were identified. Over half (15 of 26) of the urease-negative isolates (subgroups Aa and Ab) had patterns that differed only by the presence or absence of a 25-kilobase band. Urease-negative strains from other clinical material were more heterogeneous in their patterns. No correlation was apparent between strain pattern group and urease production or geographic location of isolate. The P. stuartii rDNA fingerprints were quite distinct from those of allied Providencia and Proteus species and provided a more sensitive measure of minor genomic differences than total DNA digests did.

Genomic structure and evolution of the human pepsinogen A multigene family.

A human cosmid library was screened with a pepsinogen A (PGA) cDNA probe, yielding 18 clones with (parts of) one, two or three PGA genes. By aligning these cosmids a restriction map of a PGA gene quadruplet was obtained in which the four genes are arranged in a highly ordered fashion in a head-to-tail orientation. Using the length in kilobases of the large polymorphic EcoRI fragment of the PGA genes, this quadruplet can be described as 15.0-12.0-12.0-16.6. An AvaII polymorphism allowed us to identify the two PGA haplotypes of the individual whose DNA had been cloned in the cosmid library to be a gene triplet and a gene quadruplet. By comparing the restriction maps of the central 12.0 genes in these multiplets to those of the flanking 15.0 and 16.6 genes, we postulate that these central genes arose from unequal but homologous crossing over between two 15.0-16.6 gene pairs. This hypothesis provides for the creation of a variety of haplotypes by additional cross overs and mutations. Southern blots of family and population material supports the existance of at least five common PGA haplotypes, including a single-gene haplotype, giving rise to a large number of different EcoRI patterns. The single PGA gene is probably the reciprocal crossing over product. Comparison between the DNA and protein polymorphisms suggests further micro-heterogeneity in the different PGA haplotypes.

Séquences Y-chromosome humain spécifiques présentes chez les singes anthropoïdes

We have analyzed DNAs treated with the restriction enzymes Eco RI, Taq I and Bcl I from various male and female chimpanzees and gorillas after hybridization with Y-specific human probes 49f and 49a. After Eco RI restriction, the patterns of hybridizing 49f fragments are similar between human and chimpanzee males, but differ in gorilla. Probe 49a is more Y-specific because only Y fragments are revealed, with an Eco RI pattern differing between the three species. After Taq I restriction, some 49f fragments are conserved between the chimpanzee and the human (fragments B, G, K, L, M, N, O and R); chimpanzee and man are different from other primates (the corresponding chimpanzee bands are E0, H′, J0, P′ and Q′), for polymorphic bands in human, chimpanzee is A3, C1, F1 and I1; chimpanzee is also polymorphic for bands F′, H, and one individual is AOD1. After Bcl I restriction, this same last individual is also polymorphic for bands 11-8.2 kb. Primitive alleles predicted using a previousmodel were A3, D1, F1 and I1.

Sequence heterogeneity in radish nuclear ribosomal RNA genes

The organization of nuclear rRNA genes from radish was studied. Two Bam HI fragments were cloned and used as probes in hybridization experiments. These experiments allowed to establish restriction maps for Bam HI and EcoRI sites and to demonstrate a sequence heterogeneity: two Bam HI sites are not constant and several EcoRI patterns differing by gain or loss of a site coexist. Experiments with single plants demonstrate that the observed heterogeneity is at the level of the individuals rather than the population.

Variations of chloroplast DNAs in the genus Pelargonium and their biparental inheritance

The comparison of EcoRI patterns of chloroplast DNAs (ctDNAs) from five species of the genus Pelargonium and from 16 cultivars and varieties of Pelargonium zonale hort. demonstrates a remarkable inter- and intraspecific ctDNA (plastome) variation. The plastome of the P. zonale varieties could be differentiated into groups I, II and III. Reasons for this variation seem to be: occurrence of numerous spontaneous plastome mutations, intense hybridisation by gardeners and breeders, and biparental plastid inheritance.Crosses of P. zonale varieties with different ctDNA types lead to the direct evidence on the molecular level of biparental plastid inheritance and plastid sorting-out in F1-hybrids.