The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Achilleas S Frangakis - One of the best experts on this subject based on the ideXlab platform.
-
structure of rna polymerase i transcribing Ribosomal DNA genes
Nature, 2016Co-Authors: S Neyer, Ch. Geiss, M Hantsche, Victor Valentin Hodirnau, Margot P Scheffer, Anja Seybert, Michael Kunz, Patrick Cramer, Christoph Engel, Achilleas S FrangakisAbstract:Structures of budding yeast RNA polymerase I in a catalytically active conformation are presented and confirmed by visualizing processive transcription along Ribosomal DNA genes; they support a general model for transcription elongation in which contracted and expanded polymerase conformations are associated with active and inactive states, respectively. This paper presents the long-sought structure of active, transcribing RNA polymerase I (Pol I), the enzyme that catalyses the first step in ribosome biogenesis—the transcription of Ribosomal DNA—and regulates eukaryotic cell growth. Here, Achilleas Frangakis and colleagues have determined the structures of budding yeast Pol I in a catalytically active conformation using two different techniques: cryo-electron microscopy of single particles in vitro, and cryo-electron tomography of enzymes transcribing cellular Ribosomal DNA under near-physiological conditions. The structures show that the active enzyme adopts a closed active centre conformation and support a general model for transcription elongation where contracted and expanded polymerase conformations are associated with active and inactive states, respectively. RNA polymerase I (Pol I) is a highly processive enzyme that transcribes Ribosomal DNA (rDNA) and regulates growth of eukaryotic cells1,2,3,4. Crystal structures of free Pol I from the yeast Saccharomyces cerevisiae have revealed dimers of the enzyme stabilized by a ‘connector’ element and an expanded cleft containing the active centre in an inactive conformation5,6,7. The central bridge helix was unfolded and a Pol-I-specific ‘expander’ element occupied the DNA-template-binding site. The structure of Pol I in its active transcribing conformation has yet to be determined, whereas structures of Pol II and Pol III have been solved with bound DNA template and RNA transcript8,9,10. Here we report structures of active transcribing Pol I from yeast solved by two different cryo-electron microscopy approaches. A single-particle structure at 3.8 A resolution reveals a contracted active centre cleft with bound DNA and RNA, and a narrowed pore beneath the active site that no longer holds the RNA-cleavage-stimulating domain of subunit A12.2. A structure at 29 A resolution that was determined from cryo-electron tomograms of Pol I enzymes transcribing cellular rDNA confirms contraction of the cleft and reveals that incoming and exiting rDNA enclose an angle of around 150°. The structures suggest a model for the regulation of transcription elongation in which contracted and expanded polymerase conformations are associated with active and inactive states, respectively.
Kenneth H Wilson - One of the best experts on this subject based on the ideXlab platform.
-
high density microarray of small subunit Ribosomal DNA probes
Applied and Environmental Microbiology, 2002Co-Authors: Kenneth H Wilson, W J Wilson, Jennifer L Radosevich, Todd Z Desantis, Vijay S Viswanathan, Thomas A Kuczmarski, Gary L AndersenAbstract:Ribosomal DNA sequence analysis, originally conceived as a way to provide a universal phylogeny for life forms, has proven useful in many areas of biological research. Some of the most promising applications of this approach are presently limited by the rate at which sequences can be analyzed. As a step toward overcoming this limitation, we have investigated the use of photolithography chip technology to perform sequence analyses on amplified small-subunit rRNA genes. The GeneChip (Affymetrix Corporation) contained 31,179 20-mer oligonucleotides that were complementary to a subalignment of sequences in the Ribosomal Database Project (RDP) (B. L. Maidak et al., Nucleic Acids Res. 29:173-174, 2001). The chip and standard Affymetrix software were able to correctly match small-subunit Ribosomal DNA amplicons with the corresponding sequences in the RDP database for 15 of 17 bacterial species grown in pure culture. When bacteria collected from an air sample were tested, the method compared favorably with cloning and sequencing amplicons in determining the presence of phylogenetic groups. However, the method could not resolve the individual sequences comprising a complex mixed sample. Given these results and the potential for future enhancement of this technology, it may become widely useful.
-
human colonic biota studied by Ribosomal DNA sequence analysis
Applied and Environmental Microbiology, 1996Co-Authors: Kenneth H Wilson, R B BlitchingtonAbstract:Human colonic biota is a complex microbial ecosystem that serves as a host defense. Unlike most microbial ecosystems, its composition has been studied extensively by relatively efficient culture methods. We have compared an established culture-based method with direct amplification and partial sequencing of cloned 16S rRNA genes from a human fecal specimen. Nine cycles of PCR were also compared with 35 cycles. Colonies and cloned amplicons were classified by comparing their Ribosomal DNA (rDNA; DNA coding for rRNA) sequences with rDNA sequences of known phylogeny. Quantitative culture recovered 58% of the microscopic count. The 48 colonies identified gave 21 rDNA sequences; it was estimated that 72% of the rDNA sequences from the total population of culturable cells would match these 21 sampled sequences (72% coverage). Fifty 9-cycle clones gave 27 sequences and 59% coverage of cloned rDNAs. Thirty-nine rDNAs cloned after 35 cycles of PCR gave 13 sequences for 74% coverage. Thus, the representation of the ecosystem after 35 cycles of PCR was distorted and lacked diversity. However, when the number of temperature cycles was minimized, biodiversity was preserved, and there was good agreement between culturing bacteria and sampling rDNA directly.
Gary L Andersen - One of the best experts on this subject based on the ideXlab platform.
-
high density microarray of small subunit Ribosomal DNA probes
Applied and Environmental Microbiology, 2002Co-Authors: Kenneth H Wilson, W J Wilson, Jennifer L Radosevich, Todd Z Desantis, Vijay S Viswanathan, Thomas A Kuczmarski, Gary L AndersenAbstract:Ribosomal DNA sequence analysis, originally conceived as a way to provide a universal phylogeny for life forms, has proven useful in many areas of biological research. Some of the most promising applications of this approach are presently limited by the rate at which sequences can be analyzed. As a step toward overcoming this limitation, we have investigated the use of photolithography chip technology to perform sequence analyses on amplified small-subunit rRNA genes. The GeneChip (Affymetrix Corporation) contained 31,179 20-mer oligonucleotides that were complementary to a subalignment of sequences in the Ribosomal Database Project (RDP) (B. L. Maidak et al., Nucleic Acids Res. 29:173-174, 2001). The chip and standard Affymetrix software were able to correctly match small-subunit Ribosomal DNA amplicons with the corresponding sequences in the RDP database for 15 of 17 bacterial species grown in pure culture. When bacteria collected from an air sample were tested, the method compared favorably with cloning and sequencing amplicons in determining the presence of phylogenetic groups. However, the method could not resolve the individual sequences comprising a complex mixed sample. Given these results and the potential for future enhancement of this technology, it may become widely useful.
Robin B Gasser - One of the best experts on this subject based on the ideXlab platform.
-
characterisation of ascaris from human and pig hosts by nuclear Ribosomal DNA sequences
International Journal for Parasitology, 1999Co-Authors: Neil B Chilton, D E Jacobs, Jaap Boes, Robin B GasserAbstract:Abstract The sequences of the nuclear Ribosomal DNA region spanning the first internaltranscribed spacer, the 5.8S rRNA gene and the second internal transcribed spacer weredetermined for Ascaris samples from pigs and humans from different geographicalregions. The sequences of the 5.8S gene and the second internal transcribed spacer were the samefor all samples examined, whereas all Ascaris samples from humans had six (1.3%)nucleotide differences in the first internal transcribed spacer compared with those from pigs.These differences provided some support for the existence of separate species of Ascarisor population variation within this genus. Using a nucleotide difference within a site for therestriction enzyme HaeIII, a PCR-linked restriction fragment length polymorphismmethod was established which allowed the delineation of the Ascaris samples from pigsand humans used herein. Exploiting the sequence differences in the first internal transcribedspacer, a PCR-based single-strand conformation polymorphism method was established for futureanalysis of the genetic structure of pig and human Ascaris populations in sympatric andallopatric zones. © 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd.All rights reserved.Ascaris; Internal transcribed spacer; Ribosomal DNA; Polymerasechain reaction; Restriction fragment length polymorphism; Single-strand conformationpolymorphism
-
characterisation of anisakid nematodes with zoonotic potential by nuclear Ribosomal DNA sequences
International Journal for Parasitology, 1998Co-Authors: Xingquan Zhu, Robin B Gasser, Magdalena Podolska, Neil B ChiltonAbstract:Larvae of three species of anisakid nematode from fish, Anisakis simplex, Hysterothylacium aduncum and Contracaecum osculatum, were characterised genetically using a molecular approach. The nuclear Ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S gene and the second internal transcribed spacer was amplified and sequenced. The lengths of the first and second internal transcribed spacer sequences of the three species ranged from 392 to 449 bp and 262 to 347 bp, respectively, whereas the 5.8S sequence was 157 bp. For the three species, the G+C contents for the three regions of Ribosomal DNA ranged from 42.4 to 52.2%. While no intraspecific variation was detected in the second internal transcribed spacer or 5.8S sequence of any species examined, one polymorphic nucleotide position was detected in the first internal transcribed spacer sequence for A. simplex and H. aduncum. The extent of sequence differences in the first (approximately 34-45%) and second (approximately 50-53%) internal transcribed spacers among the species was greater than in the 5.8S gene (approximately 3-5%). Based on the sequence differences, PCR-based restriction fragment length polymorphism and single-strand conformation polymorphism methods were established for the unequivocal delineation of the three species. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and population structure of each of the three anisakid nematodes examined herein, and for the diagnosis of anisakiasis in humans and animals.
-
differences in a Ribosomal DNA sequence of morphologically indistinguishable species within the hypodontus macropi complex nematoda strongyloidea
International Journal for Parasitology, 1995Co-Authors: Neil B Chilton, Robin B Gasser, Ian BeveridgeAbstract:The nucleotide sequence of the second internal transcribed spacer (ITS-2) from Ribosomal DNA has been determined for 3 members of the Hypodontus macropi species complex. Sequences were compared from nematodes collected from 3 species of Australian macropodid marsupial, Petrogale persephone, Macropus robustus robustus and Thylogale billardierii. The ITS-2 of each operational taxonomic unit ranged from 287 to 292 bases in length, and had a GC content of 36.6–40.1%. Differences in nucleotide sequence between nematodes from the different host species ranged from 25.0% to 28.3%. The data suggest that H. macropi from P. persephone represents a different species to those in M. r. robustus and T. billardierii. The unique feature of this study is that it represents a comparison of the Ribosomal DNA sequences of nematode species which are morphologically indintinguinhable but which have been demonstrated to be genetically distinct (i.e. cryptic) species based on electrophoretic data. The results also demonstrate further that morphological characters alone are often not adequate for species recognition. Differences between these 3 species of H. macropi in their recognition sites for restriction endonucleases, indicates that a PCR-RFLP approach could be used, in conjunction with allozyme electrophoresis, to establish how many species are present within the H. macropi complex.
-
differences in the second internal transcribed spacer Ribosomal DNA between five species of trichostrongylus nematoda trichostrongylidae
International Journal for Parasitology, 1995Co-Authors: Herve Hoste, Neil B Chilton, Robin B GasserAbstract:Abstract The second internal transcribed spacer (ITS-2) of the Ribosomal DNA of 5 species of Trichostrongylus has been sequenced. The ITS-2 of the 5 species was 237 or 238 bases in length, and had a GC content of approximately 30%. No evidence of intraspecific variation was detected in the ITS-2 sequence of T. colubriformis, T. vitrinus or T. retortaeformis, irrespective of the life cycle stage examined. There was evidence, however, of variation at five positions in the ITS-2 sequence of T. vitrinus samples and at one position in T. axei, indicating intra-individual variation in the sequence of different copies of the Ribosomal DNA. Nonetheless, there were consistent sequence differences between the five Trichostrongylus species examined. The level of interspecific differences in nucleotide sequence was low (1.3–7.6%), with the species infecting birds (T. tenuis) being genetically more different to the four species found in mammals. Some of the nucleotide differences between species occurred at the recognition sites of endonucleases, which makes them of important diagnostic value for species identification. Also of significance are the recognition sites for several enzymes located within the regions of sequence homology for the five species of Trichostrongylus. These may prove useful in distinguishing between genera of trichotrongyle nematodes.
Neil B Chilton - One of the best experts on this subject based on the ideXlab platform.
-
characterisation of ascaris from human and pig hosts by nuclear Ribosomal DNA sequences
International Journal for Parasitology, 1999Co-Authors: Neil B Chilton, D E Jacobs, Jaap Boes, Robin B GasserAbstract:Abstract The sequences of the nuclear Ribosomal DNA region spanning the first internaltranscribed spacer, the 5.8S rRNA gene and the second internal transcribed spacer weredetermined for Ascaris samples from pigs and humans from different geographicalregions. The sequences of the 5.8S gene and the second internal transcribed spacer were the samefor all samples examined, whereas all Ascaris samples from humans had six (1.3%)nucleotide differences in the first internal transcribed spacer compared with those from pigs.These differences provided some support for the existence of separate species of Ascarisor population variation within this genus. Using a nucleotide difference within a site for therestriction enzyme HaeIII, a PCR-linked restriction fragment length polymorphismmethod was established which allowed the delineation of the Ascaris samples from pigsand humans used herein. Exploiting the sequence differences in the first internal transcribedspacer, a PCR-based single-strand conformation polymorphism method was established for futureanalysis of the genetic structure of pig and human Ascaris populations in sympatric andallopatric zones. © 1999 Australian Society for Parasitology. Published by Elsevier Science Ltd.All rights reserved.Ascaris; Internal transcribed spacer; Ribosomal DNA; Polymerasechain reaction; Restriction fragment length polymorphism; Single-strand conformationpolymorphism
-
characterisation of anisakid nematodes with zoonotic potential by nuclear Ribosomal DNA sequences
International Journal for Parasitology, 1998Co-Authors: Xingquan Zhu, Robin B Gasser, Magdalena Podolska, Neil B ChiltonAbstract:Larvae of three species of anisakid nematode from fish, Anisakis simplex, Hysterothylacium aduncum and Contracaecum osculatum, were characterised genetically using a molecular approach. The nuclear Ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S gene and the second internal transcribed spacer was amplified and sequenced. The lengths of the first and second internal transcribed spacer sequences of the three species ranged from 392 to 449 bp and 262 to 347 bp, respectively, whereas the 5.8S sequence was 157 bp. For the three species, the G+C contents for the three regions of Ribosomal DNA ranged from 42.4 to 52.2%. While no intraspecific variation was detected in the second internal transcribed spacer or 5.8S sequence of any species examined, one polymorphic nucleotide position was detected in the first internal transcribed spacer sequence for A. simplex and H. aduncum. The extent of sequence differences in the first (approximately 34-45%) and second (approximately 50-53%) internal transcribed spacers among the species was greater than in the 5.8S gene (approximately 3-5%). Based on the sequence differences, PCR-based restriction fragment length polymorphism and single-strand conformation polymorphism methods were established for the unequivocal delineation of the three species. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and population structure of each of the three anisakid nematodes examined herein, and for the diagnosis of anisakiasis in humans and animals.
-
differences in a Ribosomal DNA sequence of morphologically indistinguishable species within the hypodontus macropi complex nematoda strongyloidea
International Journal for Parasitology, 1995Co-Authors: Neil B Chilton, Robin B Gasser, Ian BeveridgeAbstract:The nucleotide sequence of the second internal transcribed spacer (ITS-2) from Ribosomal DNA has been determined for 3 members of the Hypodontus macropi species complex. Sequences were compared from nematodes collected from 3 species of Australian macropodid marsupial, Petrogale persephone, Macropus robustus robustus and Thylogale billardierii. The ITS-2 of each operational taxonomic unit ranged from 287 to 292 bases in length, and had a GC content of 36.6–40.1%. Differences in nucleotide sequence between nematodes from the different host species ranged from 25.0% to 28.3%. The data suggest that H. macropi from P. persephone represents a different species to those in M. r. robustus and T. billardierii. The unique feature of this study is that it represents a comparison of the Ribosomal DNA sequences of nematode species which are morphologically indintinguinhable but which have been demonstrated to be genetically distinct (i.e. cryptic) species based on electrophoretic data. The results also demonstrate further that morphological characters alone are often not adequate for species recognition. Differences between these 3 species of H. macropi in their recognition sites for restriction endonucleases, indicates that a PCR-RFLP approach could be used, in conjunction with allozyme electrophoresis, to establish how many species are present within the H. macropi complex.
-
differences in the second internal transcribed spacer Ribosomal DNA between five species of trichostrongylus nematoda trichostrongylidae
International Journal for Parasitology, 1995Co-Authors: Herve Hoste, Neil B Chilton, Robin B GasserAbstract:Abstract The second internal transcribed spacer (ITS-2) of the Ribosomal DNA of 5 species of Trichostrongylus has been sequenced. The ITS-2 of the 5 species was 237 or 238 bases in length, and had a GC content of approximately 30%. No evidence of intraspecific variation was detected in the ITS-2 sequence of T. colubriformis, T. vitrinus or T. retortaeformis, irrespective of the life cycle stage examined. There was evidence, however, of variation at five positions in the ITS-2 sequence of T. vitrinus samples and at one position in T. axei, indicating intra-individual variation in the sequence of different copies of the Ribosomal DNA. Nonetheless, there were consistent sequence differences between the five Trichostrongylus species examined. The level of interspecific differences in nucleotide sequence was low (1.3–7.6%), with the species infecting birds (T. tenuis) being genetically more different to the four species found in mammals. Some of the nucleotide differences between species occurred at the recognition sites of endonucleases, which makes them of important diagnostic value for species identification. Also of significance are the recognition sites for several enzymes located within the regions of sequence homology for the five species of Trichostrongylus. These may prove useful in distinguishing between genera of trichotrongyle nematodes.
