Genetic variation between strains of the Mediterranean fruit fly, Ceratitis capitata, detected by DNA fingerprinting.

DNA fingerprinting has been used to detect genetic variation in the Mediterranean fruit fly, Ceratitis capitata. Three different probes have been identified that can be used to detect DNA restriction fragment length polymorphisms between strains of this species. The strains used in this study differ only in terms of their geographic origin or genetic background. One of the probes used is the bacteriophage vector M13, and the other two are repetitive sequences derived from the medfly genome based on a weak homology to M13. Within a strain, each probe produces a consistent restriction fragment profile that is not affected by the method or timing of DNA extraction. Between strains, when M13 is used as a probe, an average of 10% of the observable bands are polymorphic. Use of the medfly genomic sequences as a probe increases the proportion of polymorphic bands between strains up to 30%. The fact that genetic differences between even such closely related strains can be reliably detected by this method holds great promise for studies of insect pests including the ability to monitor the movements of pest species, determining the extent of genetic variation in pest populations, and in making identifications from otherwise unidentifiable material.     

M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms

Hypervariable polymorphic patterns were detected with M13 phage DNA as a probe in genomic DNA of organisms belonging to different taxonomic groups including animals (vertebrates and invertebrates), plants and microorganisms. Individual-specific restriction pattern analysis (DNA fingerprinting) with this probe proved to be useful for individual identification, analysis of somatic stability and paternity testing in man. The nuclear type of inheritance indicates that the hypervariable DNA regions in question are located in the chromosomes, not in the mitochondrial DNA. The data obtained also demonstrate a potential range of M13 DNA applications as a probe for DNA fingerprinting of animals, plants and microorganisms, particularly for the determination of inbred lines, identification of bacterial strains and establishing stock, variety and strain distinctions.     

Genomic fingerprints of organisms from different taxonomic groups: the use of phage M13 DNA as a hybridization probe

Hypervariable polymorphic patterns were detected using wild-type M13 DNA as a probe in genomic DNAs of very different organisms ranging from procaryotes and lower eucaryotes to upper plants and animals, including human beings. Due to somatic stability of highly polymorphic patterns and their discrete inheritance, individual-specific restriction pattern analysis ("DNA fingerprinting") with this test probe was found to be useful in applied human genetics, in particular, for identifying paternity and maternity, and mapping of human genomes. The data obtained also demonstrate some possibilities of the DNA fingerprinting technology in genetics and selection of agricultural plants and animals, such as variety analysis, classification and registration of individual inbred lines and strains, as well as identification of bacterial strains.     

Two regions of M13 phage genome hybridizing with human DNA are similar to several keratin genes.

DNA from two regions of the phage M13 genome hybridizes with DNA restriction fragments from genomes of various species including man [15, 20]. As the pattern of hybridization is individual-specific, this phage M13 probe can be used for DNA fingerprinting. We demonstrate here that the regions of many keratin genes coding for glycine-rich parts of C and N end domains are very similar to the phage M13 probe, and this similarity may be responsible for hybridization.     

Variability of GATA-microsatellite DNA in populations of the parthenogenetic species of lizard Lacerta unisexualis Darevsky

Multilocus DNA fingerprinting was used to analyze the genome variation of mini- and microsatellite DNA regions in parthenogenetic Caucasian rock lizard Lacerta unisexualis. The DNA fingerprints obtained with probe M13 were nearly identical in all populations examined (the average similarity index S = 0.992). The fingerprints obtained with probe (GATA)4 varied (S = 0.862). Polymorphic fragments were assumed to correspond to allelic variants of genetically unstable GATA loci. Comparison of the fingerprints of animals from four geographically isolated populations revealed several population-specific GATA microsatellite markers. Based on their distribution among the populations, the corresponding alleles were assumed to originate from a common ancestral allele.     

Genomic diversity of Oenococcus oeni from different winemaking regions of Portugal

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium that plays an important role in the elaboration of wine. It is often added as a starter culture to carry out malolactic conversion. Given the economic importance of this reaction, the taxonomic structure of this species has been studied in detail. In the present work, phenotypic and molecular approaches were used to identify 121 lactic acid bacteria strains isolated from the wines of three winemaking regions of Portugal. The strains were differentiated at the genomic level by M13-PCR fingerprinting. Twenty-seven genomic clusters represented by two or more isolates and 21 single-member clusters, based on an 85% similarity level, were recognized by hierarchic numerical analysis. M13-PCR fingerprinting patterns revealed a high level of intraspecific genomic diversity in O. oeni. Moreover, this diversity could be partitioned according to the geographical origin of the isolates. Thus, M13-PCR fingerprint analysis may be an appropriate methodology to study the O. oeni ecology of wine during malolactic fermentation as well as to trace new malolactic starter cultures and evaluate their dominance over the native microbiota.     

Genetic variation in parthenogenetic Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca, L. unisexualis) analyzed by DNA fingerprinting

Multilocus DNA fingerprinting has been used to study the variability of some mini- and microsatellite sequences in parthenogenetic species of Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca and L. unisexualis). We demonstrate that these clonally reproducing lizards possess species-specific DNA fingerprints with a low degree of intra- and interpopulation variation. Mean indices of similarity obtained using M13 DNA, (GACA)4 and (TCC)50 as probes were 0.962 and 0.966 in L. dahli and L. armeniaca, respectively. The mean index of similarity obtained using M 13 and GATA probes in L. unisexualis was estimated to be 0.95. However, despite the high degree of band-sharing, variable DNA fragments were revealed in all populations with the microsatellite probes. An particularly high level of variability was observed for (TCC)n microsatellites in populations of L. unisexualis. In fact TCC-derived DNA fingerprints were close to being individual-specific, with a mean index of similarity of 0.824. Fingerprint analysis of parthenogenetic families of L. armeniaca showed that all maternal fragments were inherited together by the progeny, and no differences in fingerprint patterns were observed. On the other hand, while identical DNA fingerprints were obtained from L. unisexualis families with M13 and (GATA)4 probes, use of the (TCC)50 probe revealed remarkable intrafamily variation in this species. It is assumed that the genetic heterogeneity observed in parthenogenetic populations may be explained, at least in part, by the existence of genetically unstable microsatellite loci. Our data serve to illustrate processes of spontaneous mutagenesis and the initial stages of clonal differentiation in natural populations of the lizard species studied.     

M13-based genotyping of marine cyanobacterial strains from the Indian subcontinent and maintained in the NFMC germplasm collection

The aim of this study was to analyze marine cyanobacterial culture collections strains of the Indian subcontinent at the level below species. This is important to improve the abilities of service culture collections to provide their user community with correctly identified and clean organisms. A total of 50 marine cyanobacterial strains were genotyped with M13 polymerase chain reaction (PCR) fingerprinting to provide diagnostic fingerprints for each culture. Depending on the strains, 9 to 26 bands were observed for the primer tested. Within the species, strains representing different isolates were genetically clearly different. Data obtained from genomic fingerprinting were used to construct binary distance matrix, and the neighbor-joining tree constructed demonstrated the ability of this method to differentiate strains at the intraspecific level. An important and useful result obtained in this study is the application of the M13 PCR fingerprinting method on almost all forms of cyanobacteria for strain and species discrimination.     

DNA fingerprints of Bombyx mori L. Testing of genotypic variability of parthenogenetic strains.

A comparative analysis of the total cellular DNA in certain parthenogenetic specimens of the silkworm (Bombyx mori), produced from the females of the parthenogenetic strains by two types of parthenogenesis, has been performed through the application of the DNA fingerprinting method based on M13 phage DNA as a hybridization probe. It has been shown that parental specimens and their genetically identical off-springs produced through ameiotic parthenogenesis have identical patterns of hybridization with the hypervariable DNA fragments. The off-springs produced through the meiotic type of parthenogenesis have individual-specific patterns of hybridization, revealing a high level of polymorphism of individual genotypes. The results obtained testify to the effectiveness and reliability of this promising method for identification of genotypic variability, marking and genomic characterization of parthenogenetic clones in the silkworm.     

Hypervariable DNA fingerprinting in Escherichia coli: minisatellite probe from bacteriophage M13.

Extensive restriction-fragment-length polymorphism was revealed in Escherichia coli strains by using a region of the bacteriophage M13 genome as a DNA hybridization probe. This variation was observed across natural strains, in clinical samples, and to a lesser extent in laboratory strains. The sequence in M13 which revealed this fingerprint pattern was a region of the gene III coat protein, which contains two clusters of a 15-base-pair repeat. Oligonucleotides made to a consensus of these repeats also revealed the fingerprint profile. While this consensus sequence has significant homology to the lambda chi site sequence, an oligonucleotide made of the chi sequence did not reveal polymorphic fingerprint patterns in E. coli. The strain variation revealed by the M13 and M13-derived oligonucleotide probes will be useful for bacterial characterization and should find use in studies of bacterial evolution and population dynamics. The findings raise questions about what these repeated sequences are and why they are so variable.     

DNA fingerprinting: a tool for determining genetic distances between strains of poultry

Summary DNA fingerprinting, a technique based on the detection of hypervariable minisatellite regions in DNA restriction fragments, was tested for its applicability to conduct population genetics in poultry. Using MspI digestion and phage M13 DNA as a probe, between 25 and 35 minisatellite-containing DNA fragments were observed per bird. Comparison of the banding pattern of offspring with their parents revealed that the bands were inherited as stable genetic traits. The variability of the DNA fingerprinting pattern was reduced in inbred strains. DNA fingerprints of chickens from five well-defined populations of known genetic relationships were analyzed and indices of genetic distances were computed. They correctly reflected the history of these strains, indicating that DNA fingerprinting may be a powerful tool to characterize genetic relationships between different breeding populations of the same species.     

Genome polymorphism in dissociative variants of Bacillus subtilis (mesentericus) 76. Identification of dissociants using genome fingerprinting

DNA fingerprinting procedure with M13 repeat probe as we have shown earlier makes it possible to apply a new approach in theoretical and applied fields of microbiology and bacteriology. In this work, using the method described we have revealed genomic polymorphism of dissociative variants of Bac. subtilis (mesentericus) 76. The data obtained may be referred as strong evidence that bacterial dissociation do has genetic nature.     

Isogenic variation of Helicobacter pylori strain resulting in heteroresistant antibacterial phenotypes in a single host in vivo

BACKGROUND: Antibiotic-susceptible and -resistant Helicobacter pylori can be present simultaneously in the same host. The aim of this study was to evaluate the genomic diversity of H. pylori strains resulting in heteroresistant antibacterial phenotypes. MATERIALS AND METHODS: Twenty-one pairs of H. pylori strains isolated from the antrum and body displaying heteroresistant antibacterial phenotypes were included. We compared the genotypes of paired-isolates by random arbitrarily primed polymerase chain reaction (PCR), flagella gene PCR-based restriction fragment length polymorphism, and flaA gene sequencing. In metronidazole-heteroresistant isolates, the sequence variation of rdxA and frxA genes was analyzed using phylogenetic analysis. RESULTS: The DNA fingerprinting patterns of the paired isolates revealed that 12 pairs (57.1%) were identical, whereas one pair (3.8%) was different. The remaining eight pairs (38.1%) of isolates showed minor heterogenecity in fingerprinting patterns. In flaA gene sequencing, these identical and similar isolates showed close sequence similarity between the antrum and body, whereas different isolate showed 31 points of different nucleotide sequences. Phylogenetic analysis of the metronidazole-heteroresistant pairs showed consistent genetic relatedness of each paired isolates despite the sequence variation of the rdxA or frxA genes in five pairs (71.4%). CONCLUSIONS: These results suggest that continuing genomic diversities in the same strain may play an important role in modulating the antibiotic-heteroresistant H. pylori in vivo.     

Fingerprinting of Mixed Bacterial Strains and BIOLOG Gram-Negative (GN) Substrate Communities by Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities. Received: 11 September 1998 / Accepted: 29 October 1998     

Differentiation of closely related and distant human populations by multilocus DNA fingerprinting

Using multilocus DNA fingerprinting with phage M13 DNA as a probe, we have investigated a heterogeneous group of four human populations from Eastern Europe and Northeastern Asia. These populations belong to two language families: Indo-European (Eastern Slavonic branch: Russians, Belarussians) and Altaian (Turkic branch: Yakuts). The experimental results were treated by different statistical techniques: cluster analysis, multidimensional scaling, and multiple correspondence analysis. Coefficients of genetic differentiation were estimated using similarity indices and heterozygosities. The results of our study demonstrated similarity of Belarussian populations and significant differences between the group of Slavonic populations and Yakuts.     

Differentiation of Closely Related and Distant Human Populations by Multilocus DNA Fingerprinting

Using multilocus DNA fingerprinting with phage M13 DNA as a probe, we have investigated a heterogeneous group of four human populations from Eastern Europe and Northeastern Asia. These populations belong to two language families: Indo-European (Eastern Slavonic branch: Russians, Belarussians) and Altaian (Turkic branch: Yakuts). The experimental results were treated by different statistical techniques: cluster analysis, multidimensional scaling, and multiple correspondence analysis. Coefficients of genetic differentiation were estimated using similarity indices and heterozygosities. The results of our study demonstrated similarity of Belarussian populations and significant differences between the group of Slavonic populations and Yakuts.     

Characterization of beta-lactam-resistant Bacteroides fragilis isolates by use of PCR fingerprinting

PCR fingerprinting was used for characterization of 35 beta-lactam-resistant Bacteroides fragilis strains isolated in Sweden and Hungary. Ten B. fragilis strains showed unique PCR fingerprints by use of the M13 core primer. Their main product was a DNA fragment with a length of 2000-bp which was absent in the other 25 strains and the reference strain B. fragilis ATCC 25285. The 2000-bp fragment from four imipenem-resistant strains gave rise to positive reactions in a specific PCR for detection of ccrA. Printed by the T3B primer, five B. fragilis strains, including the imipenem-resistant strains showed unique PCR fingerprints. The investigated imipenem-resistant strains produced carbapenem-hydrolysing metallo-beta-lactamases. The study indicates that the unique PCR fingerprinting profiles shown in highly beta-lactam resistant B. fragilis strains are correlated to antimicrobial resistance. The PCR fingerprinting technique is a useful tool for differentiation of Bacteroides fragilis strains with high-level beta-lactam resistance.     

DNA指纹图谱鉴别双歧杆菌的研究

采用RAPD技术选用10条引物对7种9株双歧杆菌基因组DNA进行PCR扩增,根据在优化条件下所得DNA指纹图谱分析了双歧杆菌菌株的遗传多样性,并构建了相似性指数矩阵和树状图.结果表明,不同序列的随机引物可扩增不同形式的RAPD图谱,但并非所有图谱都具有分类学意义,其中引物S256对双歧杆菌种及同种不同菌株均具有良好的区分能力,由该引物扩增的RAPD图谱计算出的相对性指数矩阵以及由此构建的聚类树状图均能正确地反映出双歧杆菌的系统发育关系,同时对RAPD图谱作为工业双歧杆菌分子标记的可能性进行了探讨.