The Experts below are selected from a list of 1113 Experts worldwide ranked by ideXlab platform
Guo-liang Wang - One of the best experts on this subject based on the ideXlab platform.
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Robust analysis of 5'-transcript ends: a high-throughput protocol for characterization of sequence diversity of transcription start sites.
Nature protocols, 2007Co-Authors: Malali Gowda, Guo-liang WangAbstract:The structure and diversity at the 3' ends of mRNA transcripts have been extensively characterized using several tag-based techniques in eukaryotes. However, the 5' ends of mRNA transcripts are not well understood, owing to a lack of efficient experimental approaches. We developed a new gene expression profiling method, called robust analysis of 5'-transcript ends (5' RATE), to rapidly isolate the 5' ends of mRNA transcripts. After ligating RNA oligo linkers to the 5' regions of decapped mRNA, cDNA is synthesized and digested with the restriction enzyme NlaIII. Ditags are formed by ligating two individual NlaIII tags, and are then PCR-amplified, purified and sequenced using a pyrosequencing approach. The 5'-RATE procedure is simple, fast and cost-effective because the complicated steps in comparative methods such as serial analysis of gene expression (including the formation of concatemers and their subsequent cloning and sequencing) have been eliminated. The longer 5'-RATE tags (>80 bp) provide more accurate matching to reference sequences for gene annotation and allow in-depth analysis of sequence diversity at the 5' regions of mRNA transcripts. Using our procedure, a 5'-RATE library with about 180,000 end sequences can be generated within a week. We have successfully applied the 5'-RATE method to characterize the transcriptome of various plant species including maize, rice and soybean. This method can be easily adapted to other eukaryotic organisms using the detailed procedures described in this protocol.
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Robust analysis of 5′-transcript ends (5′-RATE): a novel technique for transcriptome analysis and genome annotation
Nucleic acids research, 2006Co-Authors: Malali Gowda, Feng Chen, Joe Alessi, Richard C. Pratt, Guo-liang WangAbstract:Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes. Here we describe a new method called robust analysis of 5 0 -transcript ends (5 0 -RATE) for rapid and costeffective isolation of long 5 0 transcript ends (� 80 bp). It consists of three major steps including 5 0 -oligocapping of mRNA, NlaIII tag and ditag generation, and pyrosequencing of NlaIII tags. Complicated steps, such as purification and cloning of concatemers, colony picking and plasmid DNA purification, are eliminated and the conventional Sanger sequencing method is replaced with the newly developed pyrosequencing method. Sequence analysis of a maize 5 0 -RATE library
Malali Gowda - One of the best experts on this subject based on the ideXlab platform.
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Robust analysis of 5'-transcript ends: a high-throughput protocol for characterization of sequence diversity of transcription start sites.
Nature protocols, 2007Co-Authors: Malali Gowda, Guo-liang WangAbstract:The structure and diversity at the 3' ends of mRNA transcripts have been extensively characterized using several tag-based techniques in eukaryotes. However, the 5' ends of mRNA transcripts are not well understood, owing to a lack of efficient experimental approaches. We developed a new gene expression profiling method, called robust analysis of 5'-transcript ends (5' RATE), to rapidly isolate the 5' ends of mRNA transcripts. After ligating RNA oligo linkers to the 5' regions of decapped mRNA, cDNA is synthesized and digested with the restriction enzyme NlaIII. Ditags are formed by ligating two individual NlaIII tags, and are then PCR-amplified, purified and sequenced using a pyrosequencing approach. The 5'-RATE procedure is simple, fast and cost-effective because the complicated steps in comparative methods such as serial analysis of gene expression (including the formation of concatemers and their subsequent cloning and sequencing) have been eliminated. The longer 5'-RATE tags (>80 bp) provide more accurate matching to reference sequences for gene annotation and allow in-depth analysis of sequence diversity at the 5' regions of mRNA transcripts. Using our procedure, a 5'-RATE library with about 180,000 end sequences can be generated within a week. We have successfully applied the 5'-RATE method to characterize the transcriptome of various plant species including maize, rice and soybean. This method can be easily adapted to other eukaryotic organisms using the detailed procedures described in this protocol.
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Robust analysis of 5′-transcript ends (5′-RATE): a novel technique for transcriptome analysis and genome annotation
Nucleic acids research, 2006Co-Authors: Malali Gowda, Feng Chen, Joe Alessi, Richard C. Pratt, Guo-liang WangAbstract:Complicated cloning procedures and the high cost of sequencing have inhibited the wide application of serial analysis of gene expression and massively parallel signature sequencing for genome-wide transcriptome profiling of complex genomes. Here we describe a new method called robust analysis of 5 0 -transcript ends (5 0 -RATE) for rapid and costeffective isolation of long 5 0 transcript ends (� 80 bp). It consists of three major steps including 5 0 -oligocapping of mRNA, NlaIII tag and ditag generation, and pyrosequencing of NlaIII tags. Complicated steps, such as purification and cloning of concatemers, colony picking and plasmid DNA purification, are eliminated and the conventional Sanger sequencing method is replaced with the newly developed pyrosequencing method. Sequence analysis of a maize 5 0 -RATE library
Geraldine G. Miller - One of the best experts on this subject based on the ideXlab platform.
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Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori
Nucleic acids research, 2002Co-Authors: Richard D. Morgan, Geraldine G. Miller, Richard J. Roberts, L. J. Van Doorn, J. P. Donahue, Martin J BlaserAbstract:iceA1 in Helicobacter pylori is a homolog of NlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity.
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Genetic organization and heterogeneity of the iceA locus of Helicobacter pylori
Gene, 2000Co-Authors: Ceu Figueiredo, Ricardo Sanna, Erwin Sablon, John P. Donahue, Geraldine G. Miller, Wim Quint, Martin J Blaser, Richard M Peek, Qing Xu, Leen-jan Van DoornAbstract:Abstract The genetic organization and sequence heterogeneity of the iceA locus of Helicobacter pylori was studied, and the existence of two distinct gene families, iceA1 and iceA2, at this locus was confirmed. iceA1 has significant sequence homology to NlaIIIR, encoding an endonuclease in Neisseria lactamica, but the similarity at the protein level is limited, due to frameshift mutations of iceA1 in most H. pylori strains. In only five of the 19 iceA1 strains studied, a full-length open reading frame (ORF), capable of encoding a 228 aa protein, with 52% homology to NlaIII was observed. The region upstream of iceA2 is highly variable in length, containing up to 15 copies of 8 bp tandem repeats. iceA2 can encode proteins of 24, 59, 94, or 129 amino acids, consisting of 14 and 10 aa domains, conserved in all iceA2 strains, flanking 0, 1, 2, or 3 copies of a 35 aa cassette. This 35 aa cassette consists of domains of 13, 16 and 6 aa, respectively. The 13 aa and 6 aa domains are highly conserved, but the 16 aa domain exists in two variants. In total, five distinct iceA2 subtypes were defined. Database searches did not reveal any homologous sequences. Recombinant IceA1 and IceA2 proteins were expressed in Escherichia coli, confirming the predicted ORFs. Genotype-specific PCR primers permitted iceA genotyping in 318 (99.1%) of a worldwide collection of 321 H. pylori strains. The conserved sizes of the amplification products confirmed the worldwide distribution of discrete variants of iceA1 and iceA2.
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Analysis of iceA1 transcription in Helicobacter pylori.
Helicobacter, 2000Co-Authors: John P. Donahue, Martin J Blaser, Richard M Peek, Leen Jan Van Doorn, Stuart A. Thompson, Geraldine G. MillerAbstract:Helicobacter pylori is a Gram-negative, microaerophilic, curved bacterium that persistently colonizes the mucous layer overlying the gastric epithelium [1]. Colonization with H. pylori results in chronic superficial gastritis [2], which increases the risk for the development of duodenal and gastric ulcers, gastric adenocarcinoma, or non-Hodgkin's gastric lymphoma [3-5]. However, the majority of persons infected with H. pylori remain asymptomatic [2] and, although several strain-specific factors have been identified that are potentially markers for the differential clinical outcome of H. pylori colonization [6-9], we presently have an incomplete understanding of the bacterial factors involved in progression to ulceration or more severe disease. The iceA genetic locus was identified using an experimental strategy based on the hypothesis that adherence of H. pylori to gastric epithelial cells may induce the expression of genes related to virulence or pathogenesis [10]. DNA sequencing of iceA from H. pylori clinical isolates demonstrates that this gene exists as two distinct allelic types, designated iceA1 and iceA2 [10,11]. Epidemiological evidence suggested that strains containing the iceA1 allele are associated with duodenal ulcer disease [10,12], although this finding was not supported in another study [13]. The iceA1 allele shows significant homology (60% nucleotide identity) to NlaIIIR, which encodes the Neisseria lactamica restriction endonuclease NlaIII that recognizes the sequence CATG [14]. Located immediately downstream of iceA is hpyIM, which encodes a DNA adenine methyltransferase that also specifically recognizes the sequence CATG [15]. Consequently, the genetic arrangement of iceA1-hpyIM resembles a typical type II restriction-modification system found in many bacterial species [16]. However, the majority of iceA1 sequences studied thus far contain different, strain-specific frameshift and nonsense mutations within its potential ORF that would prevent translation of a full length protein with homology to NlaIII [10,11]. The iceA2 allele shows approximately 40% homology to iceA1 but has an entirely different genetic structure [10,11]. Thus, these observations suggest that it is unlikely that iceA-hpyIM constitutes a functional type II restriction-modification system in the majority of H. pylori strains. However, evidence for potential iceA1 function is suggested by the demonstration that iceA1 transcription is induced following H. pylori contact with epithelial cells in vitro. Consequently, we sought to characterize iceA1 transcription and determine the structure of iceA1-containing transcripts to evaluate the potential of this gene to encode functional proteins.
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The Helicobacter pylori genome is modified at CATG by the product of hpyIM.
Journal of bacteriology, 1997Co-Authors: Richard M Peek, Geraldine G. Miller, Martin J BlaserAbstract:To understand mechanisms of DNA methylation in Helicobacter pylori, a human pathogen associated with peptic ulcer disease and gastric adenocarcinoma, we cloned a putative DNA methyltransferase gene, hpyIM. This gene contains a 990-bp open reading frame encoding a 329-amino-acid protein, M.HpyI. Sequence analysis revealed that M.HpyI was closely related to CATG-recognizing adenine DNA methyltransferases, including M.NlaIII in N. lactamica. hpyIM was present in all H. pylori strains tested. DNA from wild-type H. pylori strains was resistant to digestion by SphI and NlaIII, which recognize DNA at sites containing CATG, whereas their isogenic hpyIM mutants were susceptible, indicating lack of modification. Overexpression of hpyIM in Escherichia coli rendered DNA from these cells resistant to NlaIII digestion, confirming the role of hpyIM in modifying CATG sites. We conclude that hpyIM encodes a DNA methyltransferase, M.HpyI, that is well conserved among diverse H. pylori strains and that modifies H. pylori genomes at CATG sites.
James L. Van Etten - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Chlorella virus PBCV-1 CviAII restriction and modification system
Nucleic acids research, 1992Co-Authors: Yanping Zhang, Michael Nelson, Joseph Nietfeldt, Dwight E. Burbank, James L. Van EttenAbstract:Abstract A second DNA site-specific (restriction) endonuclease (R.CviAII) and its cognate adenine DNA methyltransferase (M.CviAII) were isolated from virus PBCV-1 infected Chlorella strain NC64A cells. R.CviAII, a heteroschizomer of the bacterial restriction endonuclease NlaIII, recognizes the sequence CATG, and does not cleave CmATG sequences. However, unlike NlaIII, which cleaves after the G and does not cleave either CmATG or mCATG sequences, CviAII cleaves between the C and A and is unaffected by mCATG methylation. The M.CviAII and R.CviAII genes were cloned and their DNA sequences were determined. These genes are tandemly arranged head-to-tail such that the TAA termination codon of the M.CviAII methyltransferase gene overlaps the ATG translational start site of R.CviAII endonuclease. R.CviAII is the first chlorella virus site-specific endonuclease gene to be cloned and sequenced.
Martin J Blaser - One of the best experts on this subject based on the ideXlab platform.
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Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori
Nucleic acids research, 2002Co-Authors: Richard D. Morgan, Geraldine G. Miller, Richard J. Roberts, L. J. Van Doorn, J. P. Donahue, Martin J BlaserAbstract:iceA1 in Helicobacter pylori is a homolog of NlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity.
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Genetic organization and heterogeneity of the iceA locus of Helicobacter pylori
Gene, 2000Co-Authors: Ceu Figueiredo, Ricardo Sanna, Erwin Sablon, John P. Donahue, Geraldine G. Miller, Wim Quint, Martin J Blaser, Richard M Peek, Qing Xu, Leen-jan Van DoornAbstract:Abstract The genetic organization and sequence heterogeneity of the iceA locus of Helicobacter pylori was studied, and the existence of two distinct gene families, iceA1 and iceA2, at this locus was confirmed. iceA1 has significant sequence homology to NlaIIIR, encoding an endonuclease in Neisseria lactamica, but the similarity at the protein level is limited, due to frameshift mutations of iceA1 in most H. pylori strains. In only five of the 19 iceA1 strains studied, a full-length open reading frame (ORF), capable of encoding a 228 aa protein, with 52% homology to NlaIII was observed. The region upstream of iceA2 is highly variable in length, containing up to 15 copies of 8 bp tandem repeats. iceA2 can encode proteins of 24, 59, 94, or 129 amino acids, consisting of 14 and 10 aa domains, conserved in all iceA2 strains, flanking 0, 1, 2, or 3 copies of a 35 aa cassette. This 35 aa cassette consists of domains of 13, 16 and 6 aa, respectively. The 13 aa and 6 aa domains are highly conserved, but the 16 aa domain exists in two variants. In total, five distinct iceA2 subtypes were defined. Database searches did not reveal any homologous sequences. Recombinant IceA1 and IceA2 proteins were expressed in Escherichia coli, confirming the predicted ORFs. Genotype-specific PCR primers permitted iceA genotyping in 318 (99.1%) of a worldwide collection of 321 H. pylori strains. The conserved sizes of the amplification products confirmed the worldwide distribution of discrete variants of iceA1 and iceA2.
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Analysis of iceA1 transcription in Helicobacter pylori.
Helicobacter, 2000Co-Authors: John P. Donahue, Martin J Blaser, Richard M Peek, Leen Jan Van Doorn, Stuart A. Thompson, Geraldine G. MillerAbstract:Helicobacter pylori is a Gram-negative, microaerophilic, curved bacterium that persistently colonizes the mucous layer overlying the gastric epithelium [1]. Colonization with H. pylori results in chronic superficial gastritis [2], which increases the risk for the development of duodenal and gastric ulcers, gastric adenocarcinoma, or non-Hodgkin's gastric lymphoma [3-5]. However, the majority of persons infected with H. pylori remain asymptomatic [2] and, although several strain-specific factors have been identified that are potentially markers for the differential clinical outcome of H. pylori colonization [6-9], we presently have an incomplete understanding of the bacterial factors involved in progression to ulceration or more severe disease. The iceA genetic locus was identified using an experimental strategy based on the hypothesis that adherence of H. pylori to gastric epithelial cells may induce the expression of genes related to virulence or pathogenesis [10]. DNA sequencing of iceA from H. pylori clinical isolates demonstrates that this gene exists as two distinct allelic types, designated iceA1 and iceA2 [10,11]. Epidemiological evidence suggested that strains containing the iceA1 allele are associated with duodenal ulcer disease [10,12], although this finding was not supported in another study [13]. The iceA1 allele shows significant homology (60% nucleotide identity) to NlaIIIR, which encodes the Neisseria lactamica restriction endonuclease NlaIII that recognizes the sequence CATG [14]. Located immediately downstream of iceA is hpyIM, which encodes a DNA adenine methyltransferase that also specifically recognizes the sequence CATG [15]. Consequently, the genetic arrangement of iceA1-hpyIM resembles a typical type II restriction-modification system found in many bacterial species [16]. However, the majority of iceA1 sequences studied thus far contain different, strain-specific frameshift and nonsense mutations within its potential ORF that would prevent translation of a full length protein with homology to NlaIII [10,11]. The iceA2 allele shows approximately 40% homology to iceA1 but has an entirely different genetic structure [10,11]. Thus, these observations suggest that it is unlikely that iceA-hpyIM constitutes a functional type II restriction-modification system in the majority of H. pylori strains. However, evidence for potential iceA1 function is suggested by the demonstration that iceA1 transcription is induced following H. pylori contact with epithelial cells in vitro. Consequently, we sought to characterize iceA1 transcription and determine the structure of iceA1-containing transcripts to evaluate the potential of this gene to encode functional proteins.
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The Helicobacter pylori genome is modified at CATG by the product of hpyIM.
Journal of bacteriology, 1997Co-Authors: Richard M Peek, Geraldine G. Miller, Martin J BlaserAbstract:To understand mechanisms of DNA methylation in Helicobacter pylori, a human pathogen associated with peptic ulcer disease and gastric adenocarcinoma, we cloned a putative DNA methyltransferase gene, hpyIM. This gene contains a 990-bp open reading frame encoding a 329-amino-acid protein, M.HpyI. Sequence analysis revealed that M.HpyI was closely related to CATG-recognizing adenine DNA methyltransferases, including M.NlaIII in N. lactamica. hpyIM was present in all H. pylori strains tested. DNA from wild-type H. pylori strains was resistant to digestion by SphI and NlaIII, which recognize DNA at sites containing CATG, whereas their isogenic hpyIM mutants were susceptible, indicating lack of modification. Overexpression of hpyIM in Escherichia coli rendered DNA from these cells resistant to NlaIII digestion, confirming the role of hpyIM in modifying CATG sites. We conclude that hpyIM encodes a DNA methyltransferase, M.HpyI, that is well conserved among diverse H. pylori strains and that modifies H. pylori genomes at CATG sites.