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Hypervariable Regions (hypervariable + regions)
Selected AbstractsDevelopment and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adultsENVIRONMENTAL MICROBIOLOGY, Issue 7 2009-Stojanovi, Mirjana Rajili Summary In this paper we present the in silico assessment of the diversity of variable regions of the small subunit ribosomal RNA (SSU rRNA) gene based on an ecosystem-specific curated database, describe a probe design procedure based on two hypervariable regions with minimal redundancy and test the potential of such probe design strategy for the design of a flexible microarray platform. This resulted in the development and application of a phylogenetic microarray for studying the human gastrointestinal microbiota , referred as the human intestinal tract chip (HITChip). Over 4800 dedicated tiling oligonucleotide probes were designed based on two hypervariable regions of the SSU rRNA gene of 1140 unique microbial phylotypes (< 98% identity) following analysis of over 16 000 human intestinal SSU rRNA sequences. These HITChip probes were hybridized to a diverse set of human intestinal samples and SSU rRNA clones to validate its fingerprinting and quantification potential. Excellent reproducibility (median Pearson's correlation of 0.99) was obtained following hybridization with T7 polymerase transcripts generated in vitro from SSU rRNA gene amplicons. A linear dose,response was observed with artificial mixtures of 40 different representative amplicons with relative abundances as low as 0.1% of total microbiota. Analysis of three consecutively collected faecal samples from ten individuals (five young and five elderly adults) revealed temporal dynamics and confirmed that the adult intestinal microbiota is an individual-specific and relatively stable ecosystem. Further analysis of the stable part allowed for the identification of a universal microbiota core at the approximate genus level (90% sequence similarity). This core consists of members of Actinobacteria, Bacteroidetes and Firmicutes. Used as a phylogenetic fingerprinting tool with the possibility for relative quantification, the HITChip has the potential to bridge the gaps in our knowledge in the quantitative and qualitative description of the human gastrointestinal microbiota composition. [source] Validation of microarray-based resequencing of 93 worldwide mitochondrial genomes,HUMAN MUTATION, Issue 1 2009Anne Hartmann Abstract The human mitochondrial genome consists of a multicopy, circular dsDNA molecule of 16,569 base pairs. It encodes for 13 proteins, two ribosomal genes, and 22 tRNAs that are essential in the generation of cellular ATP by oxidative phosphorylation in eukaryotic cells. Germline mutations in mitochondrial DNA (mtDNA) are an important cause of maternally inherited diseases, while somatic mtDNA mutations may play important roles in aging and cancer. mtDNA polymorphisms are also widely used in population and forensic genetics. Therefore, methods that allow the rapid, inexpensive and accurate sequencing of mtDNA are of great interest. One such method is the Affymetrix GeneChip® Human Mitochondrial Resequencing Array 2.0 (MitoChip v.2.0) (Santa Clara, CA). A direct comparison of 93 worldwide mitochondrial genomes sequenced by both the MitoChip and dideoxy terminator sequencing revealed an average call rate of 99.48% and an accuracy of ,99.98% for the MitoChip. The good performance was achieved by using in-house software for the automated analysis of additional probes on the array that cover the most common haplotypes in the hypervariable regions (HVR). Failure to call a base was associated mostly with the presence of either a run of ,4,C bases or a sequence variant within 12 bases up- or downstream of that base. A major drawback of the MitoChip is its inability to detect insertions/deletions and its low sensitivity and specificity in the detection of heteroplasmy. However, the vast majority of haplogroup defining polymorphism in the mtDNA phylogeny could be called unambiguously and more rapidly than with conventional sequencing. Hum Mutat 0,1,8, 2008. © 2008 Wiley-Liss, Inc. [source] Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples,JOURNAL OF FORENSIC SCIENCES, Issue 3 2010Greiciane G. Paneto M.Sc. Abstract:, Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA "alleles" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2. [source] Analysis of mixed infections by multiple genotypes of human cytomegalovirus in immunocompromised patientsJOURNAL OF MEDICAL VIROLOGY, Issue 5 2009P. Sowmya Abstract Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised patients. The present study was carried out to determine the frequency of occurrence of multiple genotypes of HCMV in immunocompromised patients, to determine if there is any discrepancy in identification of mixed infections by multiple genotypes in paired clinical specimens obtained from patients and to determine the significance of viral load differences between patients infected with single and multiple genotypes. One hundred clinical specimens from 75 patients were included in the study. Real-time PCR; Multiplex PCR and PCR-based RFLP were applied for the determination of viral load and genotyping of HCMV, respectively. Out of the 75 patients, 36 (48%) carried multiple genotypes. Discrepancy with regard to detection of genotypes were found in 17/25 patients whose paired clinical specimens were analyzed. Mixed genotypes were found more often in peripheral blood than urine or intraocular fluids collected from the same patient. There was a statistically significant difference between the median viral loads of clinical specimens carrying single genotypes and multiple genotypes. Mixed infections with multiple genotypes were found predominantly in the leukocyte fraction of peripheral blood specimens. The detection of mixed infections by multiple genotypes in the hypervariable regions of HCMV can be a surrogate marker of an increase in viral load. J. Med. Virol. 81:861,869, 2009. © 2009 Wiley-Liss, Inc. [source] Refined analysis of genetic variability parameters in hepatitis C virus and the ability to predict antiviral treatment responseJOURNAL OF VIRAL HEPATITIS, Issue 8 2008J. M. Cuevas Summary., Hepatitis C virus (HCV) infects approximately 3% of the world population. The chronicity of hepatitis C seems to depend on the level of genetic variability. We have recently (Torres-Puente et al., J Viral Hepat, 2008; 15: 188) reported genetic variability estimates from a large-scale sequence analysis of 67 patients infected with HCV subtypes 1a (23 patients) and 1b (44 patients) and related them to response, or lack of, to alpha-interferon plus ribavirin treatment.. Two HCV genome regions were analysed in samples prior to antiviral therapy, one compressing the three hypervariable regions of the E2 glycoprotein and another one including the interferon sensitive determining region and the V3 domain of the NS5A protein. Haplotype and nucleotide diversity measures showed a clear tendency to higher genetic variability levels in nonresponder than in responder patients. Here, we have refined the analysis of genetic variability (haplotype and nucleotide diversity, number of haplotypes and mutations) by considering their distribution in each of the biologically meaningful subregions mentioned above, as well as in their surrounding and intervening regions. Variability levels are very heterogeneous among the different subregions, being higher for nonresponder patients. Interestingly, significant differences were detected in the biologically relevant regions, but also in the surrounding regions, suggesting that the level of variability of the whole HCV genome, rather than exclusively that from the hypervariable regions, is the main indicator of the treatment response. Finally, the number of haplotypes and mutations seem to be better discriminators than haplotype and nucleotide diversity, especially in the NS5A region. [source] E2 quasispecies specificity of hepatitis C virus association with allografts immediately after liver transplantationLIVER TRANSPLANTATION, Issue 2 2004Michael G. Hughes Jr. It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus,liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with end-stage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase-polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P = 0.03) but not at HVR2 (P = 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P = 0.02 and P = 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants. (Liver Transpl 2004;10:208,216.) [source] Variability patterns and positively selected sites at the gametophytic self-incompatibility pollen SFB gene in a wild self-incompatible Prunus spinosa (Rosaceae) populationNEW PHYTOLOGIST, Issue 3 2006Maria D. S. Nunes Summary ,,Current models for the generation of new gametophytic self-incompatibility specificities require that neutral variability segregates within specificity classes. Furthermore, one of the models predicts greater ratios of nonsynonymous to synonymous substitutions in pollen than in pistil specificity genes. All models assume that new specificities arise by mutation only. ,,To test these models, 21 SFB (the pollen S -locus) alleles from a wild Prunus spinosa (Rosaceae) population were obtained. For seven of these, the corresponding S -haplotype was also characterized. The SFB data set was also used to identify positively selected sites. Those sites are likely to be the ones responsible for defining pollen specificities. ,,Of the 23 sites identified as being positively selected, 21 are located in the variable (including a new region described here) and hypervariable regions. Little variability is found within specificity classes. There is no evidence for selective sweeps being more frequent in pollen than in pistil specificity genes. The S-RNase and the SFB genes have only partially correlated evolutionary histories. ,,None of the models is compatible with the variability patterns found in the SFB and the S -haplotype data. [source] First Genetic Insight into Libyan Tuaregs: A Maternal PerspectiveANNALS OF HUMAN GENETICS, Issue 4 2009Claudio Ottoni Summary The Tuaregs are a semi-nomadic pastoralist people of northwest Africa. Their origins are still a matter of debate due to the scarcity of genetic and historical data. Here we report the first data on the mitochondrial DNA (mtDNA) genetic characterization of a Tuareg sample from Fezzan (Libyan Sahara). A total of 129 individuals from two villages in the Acacus region were genetically analysed. Both the hypervariable regions and the coding region of mtDNA were investigated. Phylogeographic investigation was carried out in order to reconstruct human migratory shifts in central Sahara, and to shed light on the origin of the Libyan Tuaregs. Our results clearly show low genetic diversity in the sample, possibly due to genetic drift and founder effect associated with the separation of Libyan Tuaregs from an ancestral population. Furthermore, the maternal genetic pool of the Libyan Tuaregs is characterized by a major ,European" component shared with the Berbers that could be traced to the Iberian Peninsula, as well as a minor ,south Saharan' contribution possibly linked to both Eastern African and Near Eastern populations. [source] | |||||||||||||