Restriction Site Polymorphism Analysis Research Articles

The use of the hypervariable P8 region of trnL(UAA) intron for identification of orchid species: Evidence from restriction site polymorphism analysis.

The P8 stem-loop region of the trnL intron, which is known to be hypervariable in size with multiple repeat motifs and created difficulties in alignment, is always excluded in phylogenetic as well as barcode analyses. This region was investigated for species discrimination in 98 taxa of orchids belonging to the tribe Vandeae using in silico mapping of restriction site polymorphism. The length of the P8 regions varied from 200 nucleotides in Aerides rosea to 669 nucleotides in Dendrophylax sallei. Forty two taxa had unique lengths, while as many as eight shared a common length of 521 nucleotides. Of the 35 restriction endonucleases producing digestions in the P8 regions, three, viz., AgsI, ApoI and TspDTI turned out to have recognition sites across all the 98 taxa being studied. When their restriction data were combined, 92 taxa could be discriminated leaving three taxon pairs. However, Acampe papillosa and Aeranthes arachnites despite having similar restriction sites differed in their P8 lengths. This is the first report on thorough investigation of the P8 region of trnL intron for search of species specific restriction sites and hence its use as a potential plant DNA barcode.

Chloroplast DNA Variation in Some Representatives of the Asian, North American and Mediterranean Firs (Abies spp)

Abstract Using PCR-RFLP analysis, a comparative study on the restriction site polymorphism within 8 genes and regions of the Abies chloroplast DNA has been conducted covering 15 Asian, 6 North American and 7 Mediterranean species. A variable degree of divergence was observed among individual species of a given region as well as between geographical groups. A group of the Mediterranean firs, consisting of closely related species, differed profoundly from both Asian and North American representatives. Although a higher level of restriction site variants was detected among the Asian firs, two thirds of them were allocated to the difference between A. mariesii and the other Asian firs. The North American species exhibited the highest level of polymorphism resulting in several subgroups on a cladogram. At the individual species level, the Asian species A. mariesii and the North American species A. lasiocarpa diverged conspicuously from their counterparts in their respective regions. The results of restriction site polymorphism analysis are discussed with ragard to crossability and taxonomic status of individual species.

Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs

A canine distemper virus (CDV) genomic cDNA clone and expression plasmids required to establish a CDV rescue system were generated from a laboratory-adapted strain of the Onderstepoort vaccine virus. In addition, a CDV minireplicon was prepared and used in transient expression studies performed to identify optimal virus rescue conditions. Results from the transient expression experiments indicated that minireplicon-encoded reporter gene activity was increased when transfected cell cultures were maintained at 32 rather than 37 °C, and when the cellular stress response was induced by heat shock. Applying these findings to rescue of recombinant CDV (rCDV) resulted in efficient recovery of virus after transfected HEp2 or A549 cells were co-cultured with Vero cell monolayers. Nucleotide sequence determination and analysis of restriction site polymorphisms confirmed that rescued virus was rCDV. A rCDV strain also was engineered that contained the luciferase gene inserted between the P and M genes; this virus directed high levels of luciferase expression in infected cells.

Characterization of Borrelia burgdorferi isolated from erythema migrans lesions: interrelationship of three molecular typing methods.

Genetic diversity among Borrelia burgdorferi isolates recovered from the skin of Lyme disease patients was assessed by ribosomal DNA (rDNA) spacer restriction fragment length polymorphism analysis, genomic restriction site polymorphism analysis, and plasmid content analysis. There was a significant association between the three rDNA spacer types, the six pulsed-field gel types, and plasmid content (P < 0.001). The association between distinct chromosomal and plasmid markers implies a clonal origin for each genotype.

The value of rRNA gene restriction site polymorphism analysis for delineating taxa in the genus Staphylococcus.

A total of 101 staphylococcal strains were ribotyped using EcoRI and HindIII as restriction enzymes and plasmid pBA2 as the rDNA probe. Isolates from 10 newly described staphylococcal taxa were among those examined. All the ribotypes were added to our database, Staph DB, which now contains the sizes of the bands of 135 EcoRI and 120 HindIII ribotypes from 408 strains belonging to 42 staphylococcal taxa. The relatedness of ribotypes was evaluated by using the Dice coefficient. The ribotypes, and thus the strains, were clustered by the unweighted pair group method with averages (UPGMA). Separation into clusters correlated well with the delineation of the staphylococcal species but not with that of the different subspecies. No discrimination was possible between Staphylococcus vitulinus and Staphylococcus pulvereri. Ecovar-specific groups were evident within Staphylococcus intermedius and Staphylococcus hyicus. The data increase the usefulness of rRNA gene restriction site polymorphism analysis for staphylococcal taxonomy.

Suitability of Polymerase Chain Reaction-Based Approaches for Identification of Different Gypsy Moth (Lepidoptera: Lymantriidae) Genotypes in Central Europe

Two different molecular marker approaches—random amplified polymorphic DNA (RAPD) and restriction site polymorphism analysis of a polymerase chain reaction (PCR) product of the internal transcribed spacer region (ITS-2)—have been used to assess whether Asian genotypes of the gypsy moth, Lymantria dispar L., have been introduced or migrated to central Europe. In previous studies, both marker systems have proved to be reliable for distinguishing Asian and North American genotypes of this insect and thus for detecting the geographical origin of pheromone-trapped specimens in North America. RAPD analysis of >1,000 samples from 18 different geographical origins in Europe revealed that—with the exception of 2 locations—Asian, North American, and hybrid RAPD markers were present at varying proportions in all European populations. However, none of the European gypsy moth specimens was classified as Asian genotype after analysis of the ITS-2 region. The results are discussed in relation to the possible bottlenecks associated with the release of a small number of European moths in North America and the different genomic regions analyzed with both marker systems. These effects may have influenced the suitability of both diagnostics for distinguishing gypsy moth isolates from different geographical origins.

The activation of protein kinase C induces higher production of reactive oxygen species by mononuclear cells in patients with multiple sclerosis than in controls.

Recent findings have increasingly shown the importance of reactive oxygen species (ROS) in causing oxidative damage to macromolecules and in contributing to tissue degeneration in target organs of autoimmune diseases. This study was aimed at comparing the base line and induced production of ROS by peripheral blood mononuclear cells (PB MNCs) of patients with multiple sclerosis (MS) in remission and relapse, of patients with other neurological diseases (OND) and of healthy controls. In addition, we analyzed the underlying mechanism of ROS production. PB MNCs were separated from 28 MS patients in remission and 13 in relapse, and from 29 healthy controls and 10 OND. ROS was measured by spectrofluorometry. Expression of proinflammatory cytokines was assessed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Mitochondrial (mt) DNA haplotypes were determined by using restriction site polymorphism analysis. The base line and tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma induced ROS values were similar in the four groups, and the individual measures did not show a correlation with MS associated mtDNA haplotypes. Phorbol ester activation of protein kinase C (PKC) induced higher ROS production in all groups, however, with significantly greater values in the MS remission group. Calphostine C, a PKC inhibitor decreased or eliminated ROS production in a dose-dependent manner, suggesting further that it was predominantly or exclusively generated by PKC activated NADPH oxidase. A trend of increased TNF-alpha and IFN-gamma expression was noted in the MS relapse group, in contrast to the high ROS release in the MS remission group. The detected phase difference between the highest ROS production vs TNF-alpha expression is compatible with the hypothesis that different subpopulations of monocytes/macrophages are involved. We suggest that the ROS producing subpopulation preferentially migrates into the central nervous system (CNS) during a relapse. The present study together with our previous observation on oxidative damage to DNA in active plaques delineates a molecular pathway likely involved in the histologic evolution of inflammatory demyelination.

Genomic DNA restriction site heterogeneity in bovine Pasteurella multocida serogroup A isolates detected with an rRNA probe.

A total of 81 Pasteurella multocida isolates from healthy and diseased dairy and beef cattle originating from various geographical locations was examined by rRNA gene restriction site polymorphism analysis (ribotyping), restriction endonuclease analysis (REA), SDS-PAGE analysis of whole-cell (WCP) and outer-membrane (OMP) proteins, and capsule and somatic serotyping. Bacterial strains were isolated from nose, lung and in one case testicle, of Holstein and cross-bred beef cattle. The isolates represented for the most part serogroup A3 (88%). Ribotyping was performed on DNA digested with HaeII, electrophoresed and then hybridised with 32P-labelled 16S-23S rRNA from Escherichia coli. Six ribotypes (R1-R6) and 10 REA types were found among the 81 isolates with similar discrimination index (DI) of c. 0.60. Protein profiles revealed reproducibility and high levels of polymorphisms among lung isolates. Isolates were compared according to their geographical habitat, their isolation from dairy or from beef cattle and from nasal cavities or lungs. No correlation was apparent between geographical locations and ribotypes. Overall, isolates obtained from dairy cattle were predominantly R1, whereas those obtained from beef cattle were equally distributed between R1 and R2. R1 was more representative of lung isolates. For some strains, particularly the single isolate ribotypes, good correlation was achieved between WCP analysis, REA types and ribotypes. For others, REA to some extent and WCP profiles were able to discriminate among isolates within ribotypes. The data suggest that a combination of ribotyping, REA and WCP analysis is useful for investigating the epidemiology of bovine P. multocida serogroup A.

Molecular and Morphological Characters Discriminate Sitophilus oryzae and S. zeamais (Coleoptera: Curculionidae) and Confirm Reproductive Isolation

The Rice Weevil, Sitophilus oryzae (L.), and the maize weevil, S. zeamais Motschulsky, are sibling species of grain weevils that are usually distinguished by grain preferences and subtle differences in morphology. Previous findings of successful laboratory hybridization, genetic similarity of allozymes and chromosomes, and identity of aggregation pheromones raised questions about the validity of S. oryzae and S. zeamais as reproductively isolated biological species. We used molecular techniques to test the hypothesis that individuals assigned as S. oryzae or S. zeamais by morphological criteria represent members of 2 distinct gene pools, and hence are reproductively isolated species. Weevils from 18 different localities, which were collected from Africa, Australia, Asia, the south Pacific, and North America, were studied. All individuals were scored for the presence or absence of morphological characters that have been used historically for species determination. In most cases, determinations for the same individual were not consistent, depending on the morphological character used. The polymerase chain reaction (PCR) was used on the same specimens to analyze randomly amplified polymorphic DNA (RAPD-PCR) markers and to amplify selectively regions of mitochondrial DNA for analysis of restriction site polymorphisms with restriction endonucleases (RFLP-PCR). Both methods yielded markers that were consistently associated with either presence or absence of specific genitalic characters in both males and females. This correlation of molecular markers with genitalic morphotypes was consistent in all specimens studied, whether collected sympatrically from the same farms or from widely separated geographic populations, and supports a model of 2 reproductively isolated species. Other morphological characters involving pronotal punctures proved unreliable as correlates with genetic markers and are not useful for species diagnosis.

Evolutionary origins of retroposon lineages of Mhc class II Ab alleles.

Major histocompatibility complex (Mhc) class II Ab genes have evolved into three distinct lineages. While lineage 2 alleles differ from lineage 1 alleles by the insertion of a retroposon in intron 2, the basis for the extremely large intron 2 in lineage 3 alleles has heretofore been undetermined. In this report, we demonstrate by nucleotide sequencing that the genomic sequences of prototypic alleles from all three lineages diverge significantly and that lineage 3 is derived from lineage 2 by two insertional events in intron 2. One insert, composed of a member of B1 short interspersed repetitive elements (SINEs), occurs 508 base pairs (bp) 3' of exon 2, and the other, 1141 bp 3' of exon 2 within the retroposon that distinguishes lineage 2 from lineage 1. To assess the evolutionary stability of these lineages and the extent of ancestral polymorphisms of Ab within Mus species, we extended our restriction site polymorphism analysis to include 86 alleles from 120 independently derived H2 haplotypes from 12 separate species and subspecies of Mus. A phylogenetic tree revealing the relationships of these Ab alleles with respect to restriction site polymorphisms, but excluding the retroposon insertions, demonstrated that these lineages have distinctive genomic structures beyond the retroposon polymorphisms. In summary, these mouse Ab genes were produced from successive retroposon insertion events. Lineage 1 and 2 were detected in a variety of Mus species, including Mus caroli, indicating that these lineages diverged more than 2 million years ago. Lineage 3 alleles were found only in the Mus musculus subspecies, suggesting that it diverged from lineage 2 more recently. These results indicate that all three lineages of Ab have persisted through several speciation events in the genus Mus.

A genetic survey of the pathogenic fungus Ophiostoma ulmi across a Dutch elm disease front in Western Canada

The natural population structure of the Dutch elm pathogen Ophiostoma ulmi was determined from isolated collected from across a Western Canadian disease front through an analysis of restriction-site polymorphisms in the ribosomal DNA repeat, length mutations in the mitochondrial genomes, and through DNA fingerprinting of the nuclear genomes using a minisatellite DNA probe. The 8.8-kbp rDNA repeat was selected from a genomic library, and restriction-site and genic maps were constructed for the nonaggressive and aggressive subgroups of O. ulmi. There were only three restriction-site differences that distinguished these two subgroups and no intrasubgroup variation was detected. All of the isolates collected from the disease front were of the aggressive subgroup and were represented by two distinct nuclear and four mitochondrial genotypes. The minority of the isolates were of a single genotype (type A nuclear DNA; type I mtDNA), indicating the presence of a single very large clone extending across much of Manitoba and into Saskatchewan.

Genetic Diversity of Human Parvovirus B19 Determined Using a Set of Restriction Endonucleases Recognizing Four or Five Base Pairs and Partial Nucleotide Sequencing: Use of Sequence Variability in Virus Classification

Analysis of the restriction site polymorphism (RSP) of human parvovirus B19 using 12 restriction endonucleases (REs) recognizing four or five bp sequences (4- or 5-bp REs) revealed a significant difference between strains previously classified as being of the same genome type, and a relationship between two strains of different genome types, thereby indicating a global spread of B19 virus strains. These findings demonstrate the advantage of this set of 4- and 5-bp REs for the calculation of the degree of genetic diversity and clearly it is necessary to amend the taxonomy of B19 virus strains using these REs. We examined the nucleotide (nt) sequence between nt 3141 and 3411, at the N terminus of the VP2 protein coding region, in 12 B19 virus strains. The pattern of distribution of nucleotide differences between the strains confirmed the classification by RSP analysis. Between nt 3293 and nt 3364, a region in which an antigenic epitope may be encoded, there was no evidence of a nucleotide change causing an amino acid change. Thus, the amino acid sequence in this potential epitope is probably conserved.

Meiotic segregation analysis of restriction site polymorphisms allows rapid genetic mapping

The white rot fungus Phanerochaete chrysosporium is an efficient degrader of lignin. Development of classical genetics in this organism would require several years of work, so an alternative strategy was devised. As in the mapping of human genetic diseases, the approach depends on the presence of interallelic restriction site polymorphism, but differs in following their meiotic segregation into the haploid state. Using a set of 20 cloned sequences as probes we were able to demonstrate meiotic segregation of 14 restriction site polymorphisms from fruiting of a single dikaryotic culture and obtained evidence of genetic linkage within two groups of three and two of two. The method could be used to obtain a genetic map of this fungus and should be applicable to genetic analysis of any other organism for which haploid material is available.

Use of oligonucleotide hybridization in the characterization of a beta zero-thalassemia gene (beta 37 TGG----TGA) in a Saudi Arabian family [published erratum appears in Blood 1986 Jul;68(1):323

Analysis of restriction site polymorphisms in the beta-globin gene cluster of a Saudi Arabian female with beta zero-thalassemia demonstrated that both of her beta-globin genes were missing a nonpolymorphic AvaII site in exon 2. Examination of the normal nucleotide sequence surrounding this AvaII site revealed that either of two nucleotide substitutions, TGG----TAG or TGG----TGA, could produce a nonsense codon at codon 37 and eliminate the AvaII site. Consequently, two oligonucleotides (19-mers spanning codons 36 through 41 and containing either TAG or TGA at codon 37) were synthesized and hybridized against genomic DNA of the proband and her family. Specific hybridization with one of the oligomers demonstrated that the patient's beta o-thalassemia was the result of homozygosity for the TGG----TGA mutation at codon 37. In certain cases, oligonucleotide hybridization using genomic DNA may obviate the need for gene cloning and sequencing in the characterization of point mutations.

Trypanosomes of subgenus trypanozoon are diploid for housekeeping genes

The ploidy of trypanosomes has until now remained undetermined, although isoenzyme studies and direct measurements of DNA content and complexity suggest diploidy. Direct cytogenetic analysis is not possible, because the chromosomes do not condense at any stage of the cell cycle. We now present evidence from analysis of restriction site polymorphisms in and around three glycolytic enzyme genes (phosphoglycerate kinase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase) and the tubulin gene cluster, that trypanosomes of subgenus Trypanozoon are diploid for these housekeeping genes. This result is still compatible with the single copy nature of variant surface glycoprotein (VSG) genes in Trypanozoon, if different VSG genes are present in corresponding positions on paired chromosomes. Using pulse field gradient gel electrophoresis, we show that the genes for the three glycolytic enzymes are all located in very large DNA molecules, but the gene for triosephosphate isomerase is in another fraction from the genes for the other two enzymes. Since all three enzymes are located in glycosomes, which are trypanosome microbodies, the genes for glycosomal enzymes are not all clustered in one chromosomal segment of the trypanosome genome.