MboII Digestion Research Articles

Interruptions of the FXN GAA Repeat Tract Delay the Age at Onset of Friedreich's Ataxia in a Location Dependent Manner.

Friedreich’s ataxia (FRDA) is a comparatively rare autosomal recessive neurological disorder primarily caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene. The repeat expansion causes gene silencing that results in deficiency of the frataxin protein leading to mitochondrial dysfunction, oxidative stress and cell death. The GAA repeat tract in some cases may be impure with sequence variations called interruptions. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5′ and 3′ ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3′ interruptions being most frequent. Based on detection of interruptions by TP PCR assay, the patient cohort (n = 101) was stratified into four groups: 5′ interruption, 3′ interruption, both 5′ and 3′ interruptions or lacking interruption. Those patients with 3′ interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Based on this modelling, a 3′ interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5′ and 3′ interruptions. This highlights the key role of interruptions at the 3′ end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient.

Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare.

Friedreich ataxia is a multi-system autosomal recessive inherited disorder primarily caused by homozygous GAA repeat expansion mutations within intron 1 of the frataxin gene. The resulting deficiency of frataxin protein leads to progressive mitochondrial dysfunction, oxidative stress, and cell death, with the main affected sites being the large sensory neurons of the dorsal root ganglia and the dentate nucleus of the cerebellum. The GAA repeat expansions may be pure (GAA)n in sequence or may be interrupted with regions of non-GAA sequence. To our knowledge, there has been no large-scale study of FRDA patient DNA samples to determine the frequency of large interruptions in GAA repeat expansions. Therefore, we have investigated a panel of 245 Friedreich ataxia patient and carrier DNA samples using GAA repeat PCR amplification and MboII restriction enzyme digestion. We demonstrate that the vast majority (97.8%) of Friedreich ataxia GAA repeat expansion samples do not contain significant sequence changes that would result in abnormal MboII digestion profiles, indicating that they are primarily pure GAA repeats. These results show for the first time that large interruptions in the GAA repeats are very rare.

Molecular Characterization of canine parvovirus (CPV) antigenic variants from healthy and diarrheic dogs in Urmia region, Iran

Backgrounds: Canine parvovirus (CPV) has been incriminated as a primary pathogen related to acute hemorrhagic enteritis in dogs. Three major antigenic variants of CPV (CPV-2a/2b/2c) have so far been identified. Objectives: This study was carried out to investigate the frequency of CPV-2 and its variants (CPV-2a/2b/2c) in a population of healthy and diarrheic dogs in the north west of Iran. Methods: A total of 35 stool samples from healthy (n=16) and diarrheic (n=19) dogs were screened for all variants (2a, 2b, and 2c) by polymerase chain reaction (PCR) using primer pair 555for/555rev resulting in a PCR product of 583 bp in length. The resulting fragments were further digested by MboII endonuclease that selectively recognizes the restriction site “GAAGA” unique to CPV2c only. All undigested samples were subjected to PCR assays with primer pair Pab (which detects both CPV-2a and CPV-2b types) and primer pair Pb (which detect only CPV-2b type) primer pairs. The relationship of health status, breed, age, sex and vaccination status with PCR results were analyzed using statistical tests. Results: From a total of 35 samples, 10 samples were found to be positive by 555for/555rev primers that were further analyzed by MboII digestion of PCR products. One sample was characterized as CPV-2c and nine samples were categorized as CPV-2a or CPV-2b. All nine undigested samples resulted positive by PCR using Pab primers, out of which 7 resulted positive by PCR using Pb primer pairs, indicating that they are of CPV-2b variant. Conclusions: It seems that CPV-2b is prevalent variant circulating in the North West of Iran. Results also indicated that CPV-2a and CPV-2c are affecting dogs, suggests constant surveillance and monitoring of CPV variants.

Homocysteine Suppresses the Expression of the Collagen Cross-linker Lysyl Oxidase Involving IL-6, Fli1, and Epigenetic DNA Methylation

Elevated homocysteine (Hcys) serum levels represent a risk factor for several chronic pathologies, including cardiovascular disease, atherosclerosis, and chronic renal failure, and affect bone development, quality, and homeostasis. Hcys influences the formation of a stable bone matrix directly through the inhibition of the collagen cross-linking enzyme lysyl oxidase (Lox) and, as we have shown recently, by repressing its mRNA expression. The aim of this study was to investigate the mechanisms involved in this process. Through evaluation of gene arrays, quantitative RT-PCR, immunoblots, and ELISA, we identified a Hcys-dependent stimulation of interleukin 6 (IL-6) and genes involved in IL-6/Janus kinase 2 (JAK2)-dependent signal transduction pathways in pre-osteoblastic MC3T3-E1 cells. Moreover, up-regulation of genes essential for epigenetic DNA methylation (DNA (cytosine-5)-methyltransferases and helicase lymphoid-specific (Hells) was observed. Further investigations demonstrated that Hcys increased via IL-6/JAK2 the expression of Fli1 (Friend leukemia virus integration 1), a transcription factor, which we found essential for IL-6-dependent Dnmt1 stimulation. CpG methylation analysis of CpG-rich Lox proximal promoter revealed an increased CpG methylation status after treatment of the cells with Hcys indicating an epigenetic origin for Hcys-dependent Lox repression. Inhibition of the IL-6/JAK2 pathway or of CpG methylation reversed the repressive effect of Hcys on Lox expression. In conclusion, we demonstrate that Hcys stimulates IL-6 synthesis in osteoblasts, which is known to affect bone metabolism via osteoclasts. Furthermore, IL-6 stimulation results via JAK2, Fli1, and Dnmt1 in down-regulation of Lox expression by epigenetic CpG methylation revealing a new mechanism negatively affecting bone matrix formation.

Polymorphisms in the 18S rDNA gene of Cystoisospora belli and clinical features of cystoisosporosis in HIV-infected patients

Intraspecific variability among Cystoisospora belli isolates and its clinical implications in human cystoisosporosis have not been established. In this study, the restriction fragment length polymorphisms in a 1.8-kb amplicon of the small subunit ribosomal DNA (SSU rDNA) of the parasite was investigated in 20 C. belli-positive stool samples obtained from 15 HIV-infected patients. Diarrheic syndrome was observed in all patients with cystoisosporosis and the number of diarrheic episodes per patient during hospitalization ranged from 1 to 26 (mean of 9.64 ± 9.30), with a mean duration of 2 to 12 days (mean of 5.90 ± 3 days). Three restriction profiles (RF) were generated with MboII digestion, which were named RFI, RFII, and RFIII. Two isolates obtained from a patient with extraintestinal cystoisosporosis showed distinct restriction profiles with MboII. This study demonstrates that patients can be infected with different C. belli genotypes, and this information may be useful for identifying new C. belli genotypes infecting humans.

Identification of Escherichia coli O-serogroups by restrictionof the amplified O-antigen gene cluster ( rfb-RFLP)

The precise serotyping of clinical Escherichia coli isolates is a crucial step for diagnostic and epidemiological purposes. Epidemiological knowledge associated with serotyping is so important that no alternative method may be considered if it does not correlate with serotyping. Unfortunately, E. coli are difficult to serotype. Genes specifically involved in O-antigen synthesis are clustered in E. coli, Shigella and Salmonella. Published oligonucleotide sequences complementary to JUMPstart and the gnd gene (the conserved flanking sequences upstream and downstream of O-antigen gene clusters, respectively) were used to amplify the O-antigen gene cluster of representative strains of 148 E. coli O-serogroups. A unique amplified fragment was observed for each serogroup (size ranging from 1.7 to 20 kbp). Clearly identifiable and reproducible O-patterns were obtained for the great majority of O-serogroups after MboII digestion of amplified products. The number of bands composing each pattern varied from five to 25. A database was built with the patterns obtained. A total of 147 O-patterns were obtained. Thirteen O-serogroups were subdivided into different O-patterns. However, each of 13 other O-patterns was shared by two or more O-serogroups. O-serogroups of clinical isolates were deduced accurately from O-patterns in all cases, even for some rough or nonagglutinating isolates. The restriction method ( rfb-RFLP) may prove to be better than serotyping since 100% of strains are typable, which is not the case with serotyping.

Identification of Shigella serotypes by restriction of amplified O-antigen gene cluster

Due to the scarcity of distinctive biochemical reactions for differentiation of Shigella-Escherichia coli, antigenic analysis has long been used for identification and typing of Shigella isolates. Nevertheless, several intra- and interspecific cross-reactions have been reported to disturb serotyping assays. Shigella serotyping is also occasionally affected by the transition from the smooth (S) form to the rough (R) form. Thus, there is a need for the development of novel robust and discriminating methods for Shigella identification and typing. Characteristically, all genes specifically involved in O-antigen synthesis are clustered in E. coli, Shigella, and Salmonella. Published oligonucleotide sequences complementary to JUMPstart and gene gnd, the conserved flanking sequences upstream and downstream of O-antigen gene clusters, were used to amplify the O-antigen gene cluster of representative strains of each Shigella serotype. A unique, amplified fragment was generally observed for each serotype (size ranging from 6 kbp to 17 kbp). Clearly identifiable and reproducible patterns were obtained for each serotype after MboII digestion of the products, except for S. boydii 12 which showed two distinct patterns, and S. flexneri serotypes 1 to 5 and X and Y which showed a single pattern. A database was built with the Taxotron package allowing automated identification of clinical Shigella isolates to all known serotypes.

Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure

BACKGROUNDIt was recently reported that A to G transition mutations at positions 2143 and 2144 in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori.AIMSTo study the...

Characterization of the rpsL and rrs genes of streptomycin‐resistant clinical isolates of Mycobacterium tuberculosis in Japan

Mutations in the rpsL and rrs genes associated with streptomycin resistance in Mycobacterium tuberculosis clinically isolated in Japan were characterized. The rpsL genes of 172 clinical isolates were amplified by PCR and classified into two groups on the basis of MboII restriction digestion. Thirty-three out of 54 (61.1%) streptomycin-highly resistant isolates (MIC > 200 micrograms ml-1) were not digested by MboII. By contrast, the remaining 21 of 54 (38.9%) streptomycin-highly resistant isolates, all of 41 isolates with streptomycin resistance at a lower level (20 micrograms ml-1 < MIC < or = 200 micrograms ml-1), and all of 77 streptomycin-sensitive isolates, were restricted. Thus, all isolates resistant for MboII digestion showed a high level of resistance to streptomycin. Subsequently, the sequence for the rpsL and rrs genes from the 46 isolates were analysed. Eighteen out of 19 (94.7%) streptomycin-highly resistant isolates carried a mutation in any rpsL gene at position 43 or 88, or the rrs gene; 10 out of 17 (58.8%) streptomycin-resistant isolates at a lower level were confirmed to exhibit the mutation of either the mutated rpsL gene at position 88, or the rrs gene. In the total 36 streptomycin-resistant isolates, the mutation of the rpsL or rrs gene was observed in 28 streptomycin-resistant isolates, corresponding to 77.8%, whereas none of the streptomycin-sensitive isolates had mutations in either the rpsL or rrs gene.

Recognition of two new species of intestinal spirochetes: Serpulina intermedia sp. nov. and Serpulina murdochii sp. nov.

On the basis of DNA-DNA hybridization data, nine intestinal spirochete strains were grouped into five genospecies. Three of these genospecies were previously recognized Serpulina species, Serpulina hyodysenteriae (type strain, B78), Serpulina innocens (type strain, B256), and Serpulina pilosicoli (type strain, P43/6/78; previously "Anguillina coli"). The other two genospecies were found to be new Serpulina species, for which we propose the names Serpulina intermedia sp. nov. (with type strain PWS/A) and Serpulina murdochii sp. nov. (with type strain 56-150). S. intermedia and S. murdochii cells had a typical spirochete ultrastructure with 22 to 28 periplasmic flagella per cell. Various soluble sugars were growth substrates for S. intermedia and S. murdochii. During growth in basal heart infusion broth supplemented with fetal calf serum beneath an O2-N2 (1:99) atmosphere, cells of these new species consumed oxygen and glucose and produced H2, CO2, acetate, butyrate, and ethanol. The G + C content of the DNA of S. murdochii 56-150T was 27 mol%, and the G + C content of the DNA of S. intermedia PWS/AT was 25 mol%. In addition, a restriction fragment length polymorphism-PCR assay for the detection of intestinal spirochetes was developed. The assay was based on generation and restriction endonuclease analysis (with HinfI, TaqI, Sau3A, and MboII) of a 558-bp amplicon of ribosomal DNA (rDNA) encoding 16S rRNA. The PCR amplification was specific for Serpulina species and Brachyspira aalborgi. Four restriction digest patterns were found for the five Serpulina species. HinfI restriction differentiated S. murdochii and S. innocens from the other species. Sau3A and TaqI restrictions gave unique fragment patterns for S. murdochii and S. pilosicoli, respectively. S. hyodysenteriae and S. intermedia DNAs gave the same fragment pattern regardless of the enzyme tested. B. aalborgi was differentiated from the Serpulina species by MboII digestion of the 16S rDNA amplicon.