The Experts below are selected from a list of 288 Experts worldwide ranked by ideXlab platform
Michael Mcclelland - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the SOS Response in Salmonella enterica Serovar Typhimurium Using RNA Fingerprinting by Arbitrarily Primed PCR
Journal of bacteriology, 2000Co-Authors: Nicholas R. Benson, Rita M.-y. Wong, Michael McclellandAbstract:We report an analysis of a sample of the SOS response of Salmonella enterica serovar Typhimurium using the differential display of RNA Fingerprinting gels of arbitrarily primed PCR products. The SOS response was induced by the addition of mitomycin C to an exponentially growing culture of serovar Typhimurium, and the RNA population was sampled during the following 2 h. These experiments revealed 21 differentially expressed PCR fragments representing mRNA transcripts. These 21 fragments correspond to 20 distinct genes. All of these transcripts were positively regulated, with the observed induction starting 10 to 120 min after addition of mitomycin C. Fifteen of the 21 transcripts have no homologue in the public sequence data banks and are therefore classified as novel. The remaining six transcripts corresponded to the recE, stpA, sulA, and umuC genes, and to a gene encoding a hypothetical protein in the Escherichia coli lysU-cadA intergenic region; the recE gene was represented twice by nonoverlapping fragments. In order to determine if the induction of these 20 transcripts constitutes part of a classical SOS regulon, we assessed the induction of these genes in a recA mutant. With one exception, the increased expression of these genes in response to mitomycin C was dependent on the presence of a functional recA allele. The exception was fivefold induced in the absence of a functional RecA protein, suggesting another layer of regulation in response to mitomycin C, in addition to the RecA-LexA pathway of SOS induction. Our data reveal several genes belonging to operons known to be directly involved in pathogenesis. In addition, we have found several phage-like sequences, some of which may be landmarks of pathogenicity determinants. On the basis of these observations, we propose that the general use of DNA-damaging agents coupled with differential gene expression analysis may be a useful and easy method for identifying pathogenicity determinants in diverse organisms.
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Screening differentially displayed PCR products by single-strand conformation polymorphism gels
PCR Applications, 1999Co-Authors: Françoise Mathieu-daudé, Michael Mcclelland, Nick Benson, Frank Kullmann, Rhonda J. Honeycutt, John WelshAbstract:Publisher Summary Differential display and ribonucleic acid (RNA) arbitrarily primed PCR have been extensively used to investigate differential gene expression and coordinate regulation in a wide variety of situations, including cell responses to different treatments and growth conditions and comparisons of different developmental stages. The RNA Fingerprinting approach has also found many applications in cancer research. Several modifications to the original protocols have been reported, and the development of new technologies, such as automated sequencing or the use of fluorescent-tagged primers, is facilitating the use of this approach RNA Fingerprinting has three steps: (i) cDNA synthesis, (ii) isolation and characterization of differentially amplified transcripts, and (iii) confirmation of differential expression. The step of isolation and characterization of the PCR fragments representing differentially amplified products is often a problem. This is so because it's laborious or gives products that are not differentially expressed because they are not the ones being targeted. These problems are associated with the comigration of other PCR products in the fingerprint gel. This chapter focuses on the use of single-strand polymorphism gels to facilitate the isolation and purification of differentially amplified cDNAs from differential display and RAP-PCR Fingerprinting experiments. The two protocols that are currently in use are also provided.
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Identification of differentially expressed genes using RNA Fingerprinting by arbitrarily primed polymerase chain reaction.
Methods in enzymology, 1999Co-Authors: Françoise Mathieu-daudé, John Welsh, Thomas Vogt, Barbara H. Jung, Thomas Trenkle, Michael McclellandAbstract:Publisher Summary Genomic Fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) is a sensitive and efficient method for generating a large number of molecular markers. Based on the selective amplification of DNA sequences flanked, by chance, by sequences matching an arbitrarily chosen primer, AP-PCR reveals sequence polymorphisms between different template DNAs. The ability to detect differences between fingerprints of closely related organisms made this approach a useful tool for studying genetic diversity, population biology, epidemiology, and genetic mapping. The more recent application of AP-PCR Fingerprinting to RNA, differential display, or RNA-arbitrarily primed PCR (RAP-PCR) has resulted in an interesting tool for the detection of differential gene expression. This chapter focuses on the use of RAP-PCR Fingerprinting. It describes the method and provides the present protocols for the different steps from initial RNA preparation through sequence analysis and confirmation of differential expression of the transcripts identified.
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Differentially expressed genes in the Trypanosoma brucei life cycle identified by RNA Fingerprinting.
Molecular and biochemical parasitology, 1998Co-Authors: Françoise Mathieu-daudé, John Welsh, Charles E. Davis, Michael McclellandAbstract:Abstract RNA Fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify genes that were differentially expressed during the life cycle of Trypanosoma brucei , as well as in response to heat shock. The standard RAP-PCR protocol was varied in two ways. First, the PCR reactions sometimes included a primer derived from the 5′ mini-exon sequence, to ensure that most of the products contained the 5′ end of mRNAs. Second, differentially amplified products were reamplified, isolated on single strand conformation polymorphism (SSCP) gels, cloned, and sequenced. Clones representing 32 different expressed sequence tags (ESTs) were obtained. Twenty-four ESTs were confirmed as differentially expressed by RT-PCR between different stages of the parasite cycle, or in response to temperature elevation. Nine clones had significant similarities to sequences already in the database. These transcripts included genes encoding cell surface proteins, metabolic enzymes, and heat shock proteins, either from trypanosomes or other organisms. Of particular interest, ESAG1 was shown to be heat-inducible in the procyclic stage. Most of the transcripts were unrelated to any other sequences in the database, and were deposited as new ESTs. The identification of stage-specific and heat shock-regulated transcripts will complement the growing T. brucei database. In addition, this experimental approach allows previous entries in the sequence database to be annotated with regulatory information.
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Applications of DNA and RNA Fingerprinting by the Arbitrarily Primed Polymerase Chain Reaction
Bacterial Genomes, 1998Co-Authors: Françoise Mathieu-daudé, John Welsh, Frank Kullmann, Rhonda J. Honeycutt, Thomas Vogt, Karen Evans, Michael McclellandAbstract:The related methods Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) and Random Amplified Polymorphic DNA (RAPD) were developed independently by our group (Welsh and McClelland, 1990) and by Williams et al. (1990). These methods were based on the observation that PCR performed at relatively low stringency (in the case of AP-PCR) or with low selectivity primers at high stringency (in the case of RAPD) yield a reproducible collection of products that depend on the template and primer sequences. Arrayed on an agarose or acry1-amide gel, this collection of products can be viewed as a “fingerprint” or “bar code” for the DNA template. Because of their dependence on template sequence, AP-PCR and RAPD can be used as methods for sampling in sequence space; diverse applications can be imagined, many of which have been demonstrated in the literature.
John Welsh - One of the best experts on this subject based on the ideXlab platform.
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Identification of differentially expressed genes using RNA Fingerprinting by arbitrarily primed polymerase chain reaction.
Methods in enzymology, 1999Co-Authors: Françoise Mathieu-daudé, John Welsh, Thomas Vogt, Barbara H. Jung, Thomas Trenkle, Michael McclellandAbstract:Publisher Summary Genomic Fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) is a sensitive and efficient method for generating a large number of molecular markers. Based on the selective amplification of DNA sequences flanked, by chance, by sequences matching an arbitrarily chosen primer, AP-PCR reveals sequence polymorphisms between different template DNAs. The ability to detect differences between fingerprints of closely related organisms made this approach a useful tool for studying genetic diversity, population biology, epidemiology, and genetic mapping. The more recent application of AP-PCR Fingerprinting to RNA, differential display, or RNA-arbitrarily primed PCR (RAP-PCR) has resulted in an interesting tool for the detection of differential gene expression. This chapter focuses on the use of RAP-PCR Fingerprinting. It describes the method and provides the present protocols for the different steps from initial RNA preparation through sequence analysis and confirmation of differential expression of the transcripts identified.
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Screening differentially displayed PCR products by single-strand conformation polymorphism gels
PCR Applications, 1999Co-Authors: Françoise Mathieu-daudé, Michael Mcclelland, Nick Benson, Frank Kullmann, Rhonda J. Honeycutt, John WelshAbstract:Publisher Summary Differential display and ribonucleic acid (RNA) arbitrarily primed PCR have been extensively used to investigate differential gene expression and coordinate regulation in a wide variety of situations, including cell responses to different treatments and growth conditions and comparisons of different developmental stages. The RNA Fingerprinting approach has also found many applications in cancer research. Several modifications to the original protocols have been reported, and the development of new technologies, such as automated sequencing or the use of fluorescent-tagged primers, is facilitating the use of this approach RNA Fingerprinting has three steps: (i) cDNA synthesis, (ii) isolation and characterization of differentially amplified transcripts, and (iii) confirmation of differential expression. The step of isolation and characterization of the PCR fragments representing differentially amplified products is often a problem. This is so because it's laborious or gives products that are not differentially expressed because they are not the ones being targeted. These problems are associated with the comigration of other PCR products in the fingerprint gel. This chapter focuses on the use of single-strand polymorphism gels to facilitate the isolation and purification of differentially amplified cDNAs from differential display and RAP-PCR Fingerprinting experiments. The two protocols that are currently in use are also provided.
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Differentially expressed genes in the Trypanosoma brucei life cycle identified by RNA Fingerprinting.
Molecular and biochemical parasitology, 1998Co-Authors: Françoise Mathieu-daudé, John Welsh, Charles E. Davis, Michael McclellandAbstract:Abstract RNA Fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify genes that were differentially expressed during the life cycle of Trypanosoma brucei , as well as in response to heat shock. The standard RAP-PCR protocol was varied in two ways. First, the PCR reactions sometimes included a primer derived from the 5′ mini-exon sequence, to ensure that most of the products contained the 5′ end of mRNAs. Second, differentially amplified products were reamplified, isolated on single strand conformation polymorphism (SSCP) gels, cloned, and sequenced. Clones representing 32 different expressed sequence tags (ESTs) were obtained. Twenty-four ESTs were confirmed as differentially expressed by RT-PCR between different stages of the parasite cycle, or in response to temperature elevation. Nine clones had significant similarities to sequences already in the database. These transcripts included genes encoding cell surface proteins, metabolic enzymes, and heat shock proteins, either from trypanosomes or other organisms. Of particular interest, ESAG1 was shown to be heat-inducible in the procyclic stage. Most of the transcripts were unrelated to any other sequences in the database, and were deposited as new ESTs. The identification of stage-specific and heat shock-regulated transcripts will complement the growing T. brucei database. In addition, this experimental approach allows previous entries in the sequence database to be annotated with regulatory information.
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Applications of DNA and RNA Fingerprinting by the Arbitrarily Primed Polymerase Chain Reaction
Bacterial Genomes, 1998Co-Authors: Françoise Mathieu-daudé, John Welsh, Frank Kullmann, Rhonda J. Honeycutt, Thomas Vogt, Karen Evans, Michael McclellandAbstract:The related methods Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) and Random Amplified Polymorphic DNA (RAPD) were developed independently by our group (Welsh and McClelland, 1990) and by Williams et al. (1990). These methods were based on the observation that PCR performed at relatively low stringency (in the case of AP-PCR) or with low selectivity primers at high stringency (in the case of RAPD) yield a reproducible collection of products that depend on the template and primer sequences. Arrayed on an agarose or acry1-amide gel, this collection of products can be viewed as a “fingerprint” or “bar code” for the DNA template. Because of their dependence on template sequence, AP-PCR and RAPD can be used as methods for sampling in sequence space; diverse applications can be imagined, many of which have been demonstrated in the literature.
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RNA Fingerprinting Displays UVB-specific Disruption of Transcriptional Control in Human Melanocytes
Cancer research, 1997Co-Authors: Thomas Vogt, John Welsh, Frank Kullmann, Wilhelm Stolz, Barbara H. Jung, Michael Landthaler, Michael McclellandAbstract:In mammalian cells, UV induces a limited set of early transcribed genes, which overlaps with the set of genes induced by tumor promoting drugs such as 12-O-tetradecanoyl phorbol-13-acetate (TPA). Among these are genes for transcription factors, the activation of which leads to complex secondary changes in expression of multiple target genes. How these delayed pleiotropic UV effects on transcription may contribute to initiation of melanoma skin cancer is poorly understood. We analyzed changes in the relative abundances of 1900 transcripts in newborn human melanocytes 8 h after treatment with UVB, TPA, and cycloheximide in all combinations, using RNA arbitrarily primed PCR for differential display. The relative abundances of 205 transcripts (11% of all transcripts surveyed) were altered by one or more of the treatment combinations. Fourteen of the 77 genes up-regulated by TPA were also up-regulated by UVB, but 60 of the TPA up-regulated genes were down-regulated by UVB, indicating both intersecting and independent signal transduction pathways for UVB and TPA. A number of UVB and TPA target genes were identified by cDNA cloning. Consistent with UVB induction of a partly transformed phenotype in mammalian cells, UVB antagonized the TPA-inducible expression of tumor-suppressive tropomyosin 3 mRNA. In addition, UVB may impair mitochondrial functioning and induce oxidative stress by strong down-regulation of mitochondrial transcription. Finally, increased expression of the dihydropteridine reductase gene, a major regulator of the cellular tetrahydrobiopterin pool, was linked to the UV pathway.
Kulvinder S. Gill - One of the best experts on this subject based on the ideXlab platform.
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Identification of Wheat Chromosomal Regions Containing Expressed Resistance Genes
Genetics, 2004Co-Authors: Muharrem Dilbirligi, Mustafa Erayman, Devinder Sandhu, Deepak Sidhu, Kulvinder S. GillAbstract:The objectives of this study were to isolate and physically localize expressed resistance (R) genes on wheat chromosomes. Irrespective of the host or pest type, most of the 46 cloned R genes from 12 plant species share a strong sequence similarity, especially for protein domains and motifs. By utilizing this structural similarity to perform modified RNA Fingerprinting and data mining, we identified 184 putative expressed R genes of wheat. These include 87 NB/LRR types, 16 receptor-like kinases, and 13 Pto-like kinases. The remaining were seven Hm1 and two Hs1(pro-1) homologs, 17 pathogenicity related, and 42 unique NB/kinases. About 76% of the expressed R-gene candidates were rare transcripts, including 42 novel sequences. Physical mapping of 121 candidate R-gene sequences using 339 deletion lines localized 310 loci to 26 chromosomal regions encompassing approximately 16% of the wheat genome. Five major R-gene clusters that spanned only approximately 3% of the wheat genome but contained approximately 47% of the candidate R genes were observed. Comparative mapping localized 91% (82 of 90) of the phenotypically characterized R genes to 18 regions where 118 of the R-gene sequences mapped.
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Identification and analysis of expressed resistance gene sequences in wheat
Plant Molecular Biology, 2003Co-Authors: Muharrem Dilbirligi, Kulvinder S. GillAbstract:Forty-eight resistance ( R ) genes conferring resistance to various types of pests have been cloned from 12 plant species. Irrespective of the host or the pest type, most R genes share a strong protein sequence similarity especially for domains and motifs. The objective of this study was to identify expressed R genes of wheat, the fraction of which is expected to be very low in the genome. Using modified RNA Fingerprinting and data mining approaches we identified 220 expressed R -gene candidates. Of these, 125 sequences structurally resembled known R genes. In addition to 25–87% protein sequence similarity with the known R genes, the sequence, order, and distribution of the domains and motifs were also the same. Among the remaining 95, 17 were probable R -related, 21 were a new class of nucleotide-binding kinases, 21 were probable kinases, and 36 were p-loop-containing unknown sequences. About 76% were rare including 73 novel sequences. Three new R- gene specific motifs were also identified. Physical mapping of the 164 best R -gene candidates on 339 deletion lines localized 121 mappable R -gene candidates to 26 small chromosomal regions encompassing about 16% of the genome. About 90 of the 110 phenotypically characterized wheat R genes corresponding to 18 different pests also mapped in these regions.
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Identification of expressed sequence markers for a major gene-rich region of wheat chromosome group 1 using RNA Fingerprinting-differential display
Crop Science, 2002Co-Authors: Devinder Sandhu, Deepak Sidhu, Kulvinder S. GillAbstract:This study demonstrates a successful application of RNA Fingerprinting-differential display technique in identifying expressed sequence markers for a small targeted region of the wheat (Triticum aestivum L.) genome. Wheat genes are present in clusters spanning about 10% of the genome. One of the important gene-rich regions is present on the short arm of wheat homoeologous group I chromosomes around fraction length 0.8 ('1S0.8 region'). The region is about 0.1% of the wheat genome and is flanked by the breakpoints of deletion lines 1BS-4 and 1BS-19. The objective of this study was to identify expressed sequence markers for the region. First-strand cDNA of poly A + mRNA pooled from various developmental stages of the two deletion lines were PCR amplified in the presence of 35 S by means of 90 pair-wise combinations of 19 primers. Amplification products were size-separated on a denaturing polyacrylamide urea gel. A total of 6840 fragment bands were amplified, of which 65 were present in the deletion line 1BS-4, but missing in 1BS-19. These 65 fragment bands were cut out of the gel, reamplified, and used as probes for gel-blot DNA analysis of group 1 nullisomic-tetrasomic lines and the two deletion lines. Nineteen of the 65 fragment bands detected a smear pattern and thus were not mapped. Of the remaining 46 probes, 22 mapped to wheat homoeologous group 1 and seven mapped to the '1S0.8 region'. The same approach can be used to target other wheat gene-rich regions bracketed by deletion breakpoints.
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Candidate-gene cloning and targeted marker enrichment of wheat chromosomal regions using RNA Fingerprinting - differential display
Genome, 2001Co-Authors: Kulvinder S. Gill, Devinder SandhuAbstract:The usefulness of the RNA Fingerprinting - differential display technique in gene cloning and targeted marker enrichment in wheat is demonstrated. A small region of chromosome 5BL was targeted that contains Ph1 ,a chromosome-pairing regulator gene. The cultivar Chinese Spring (CS) and mutant ph1b are almost identical except for chromosome 5BL, which, in the mutant line, carries an interstitial deletion encompassing the Ph1 gene. Poly(A) + RNA of the two lines from anthers at developmental stages ranging from pre-meiotic mitosis to anaphase II was PCR- amplified using 38 pairwise combinations of 19 primers. The 35 S-labeled amplified products were size-separated on de- naturing polyacrylamide-urea gels. A total of 3154 fragment bands were observed, of which 43 were present in CS but absent in the ph1b mutant. These 43 fragment bands were eluted, re-amplified, and used as probes in gel-blot DNA analyses of wheat group 5 nullisomic-tetrasomic lines and the ph1b mutant. Twenty-four of these 43 probes were sin- gle- or few-copy sequences. Eight of the 24 probes mapped to wheat group 5 and five mapped to the deletion of the ph1b mutant. Three of these five probes were further localized to the submicroscopic region containing the Ph1 gene, by using two deletion lines flanking the region. Northern-blot analysis revealed that the gene corresponding to one of these three probes expresses mainly during meiosis and is from the B genome.
Françoise Mathieu-daudé - One of the best experts on this subject based on the ideXlab platform.
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Screening differentially displayed PCR products by single-strand conformation polymorphism gels
PCR Applications, 1999Co-Authors: Françoise Mathieu-daudé, Michael Mcclelland, Nick Benson, Frank Kullmann, Rhonda J. Honeycutt, John WelshAbstract:Publisher Summary Differential display and ribonucleic acid (RNA) arbitrarily primed PCR have been extensively used to investigate differential gene expression and coordinate regulation in a wide variety of situations, including cell responses to different treatments and growth conditions and comparisons of different developmental stages. The RNA Fingerprinting approach has also found many applications in cancer research. Several modifications to the original protocols have been reported, and the development of new technologies, such as automated sequencing or the use of fluorescent-tagged primers, is facilitating the use of this approach RNA Fingerprinting has three steps: (i) cDNA synthesis, (ii) isolation and characterization of differentially amplified transcripts, and (iii) confirmation of differential expression. The step of isolation and characterization of the PCR fragments representing differentially amplified products is often a problem. This is so because it's laborious or gives products that are not differentially expressed because they are not the ones being targeted. These problems are associated with the comigration of other PCR products in the fingerprint gel. This chapter focuses on the use of single-strand polymorphism gels to facilitate the isolation and purification of differentially amplified cDNAs from differential display and RAP-PCR Fingerprinting experiments. The two protocols that are currently in use are also provided.
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Identification of differentially expressed genes using RNA Fingerprinting by arbitrarily primed polymerase chain reaction.
Methods in enzymology, 1999Co-Authors: Françoise Mathieu-daudé, John Welsh, Thomas Vogt, Barbara H. Jung, Thomas Trenkle, Michael McclellandAbstract:Publisher Summary Genomic Fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) is a sensitive and efficient method for generating a large number of molecular markers. Based on the selective amplification of DNA sequences flanked, by chance, by sequences matching an arbitrarily chosen primer, AP-PCR reveals sequence polymorphisms between different template DNAs. The ability to detect differences between fingerprints of closely related organisms made this approach a useful tool for studying genetic diversity, population biology, epidemiology, and genetic mapping. The more recent application of AP-PCR Fingerprinting to RNA, differential display, or RNA-arbitrarily primed PCR (RAP-PCR) has resulted in an interesting tool for the detection of differential gene expression. This chapter focuses on the use of RAP-PCR Fingerprinting. It describes the method and provides the present protocols for the different steps from initial RNA preparation through sequence analysis and confirmation of differential expression of the transcripts identified.
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Differentially expressed genes in the Trypanosoma brucei life cycle identified by RNA Fingerprinting.
Molecular and biochemical parasitology, 1998Co-Authors: Françoise Mathieu-daudé, John Welsh, Charles E. Davis, Michael McclellandAbstract:Abstract RNA Fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify genes that were differentially expressed during the life cycle of Trypanosoma brucei , as well as in response to heat shock. The standard RAP-PCR protocol was varied in two ways. First, the PCR reactions sometimes included a primer derived from the 5′ mini-exon sequence, to ensure that most of the products contained the 5′ end of mRNAs. Second, differentially amplified products were reamplified, isolated on single strand conformation polymorphism (SSCP) gels, cloned, and sequenced. Clones representing 32 different expressed sequence tags (ESTs) were obtained. Twenty-four ESTs were confirmed as differentially expressed by RT-PCR between different stages of the parasite cycle, or in response to temperature elevation. Nine clones had significant similarities to sequences already in the database. These transcripts included genes encoding cell surface proteins, metabolic enzymes, and heat shock proteins, either from trypanosomes or other organisms. Of particular interest, ESAG1 was shown to be heat-inducible in the procyclic stage. Most of the transcripts were unrelated to any other sequences in the database, and were deposited as new ESTs. The identification of stage-specific and heat shock-regulated transcripts will complement the growing T. brucei database. In addition, this experimental approach allows previous entries in the sequence database to be annotated with regulatory information.
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Applications of DNA and RNA Fingerprinting by the Arbitrarily Primed Polymerase Chain Reaction
Bacterial Genomes, 1998Co-Authors: Françoise Mathieu-daudé, John Welsh, Frank Kullmann, Rhonda J. Honeycutt, Thomas Vogt, Karen Evans, Michael McclellandAbstract:The related methods Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) and Random Amplified Polymorphic DNA (RAPD) were developed independently by our group (Welsh and McClelland, 1990) and by Williams et al. (1990). These methods were based on the observation that PCR performed at relatively low stringency (in the case of AP-PCR) or with low selectivity primers at high stringency (in the case of RAPD) yield a reproducible collection of products that depend on the template and primer sequences. Arrayed on an agarose or acry1-amide gel, this collection of products can be viewed as a “fingerprint” or “bar code” for the DNA template. Because of their dependence on template sequence, AP-PCR and RAPD can be used as methods for sampling in sequence space; diverse applications can be imagined, many of which have been demonstrated in the literature.
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RNA Fingerprinting and differential display using arbitrarily primed PCR
Trends in genetics : TIG, 1995Co-Authors: Michael Mcclelland, Françoise Mathieu-daudé, John WelshAbstract:RNA Fingerprinting by arbitrarily primed PCR can be used to detect and clone transcripts that are differentially expressed between cells that have been subject to different environments or developmental programs. The method also allows an estimate of the number of genes that are differentially expressed under various circumstances. When many experimental treatments are compared in parallel, intersecting regulatory pathways are reflected in genes that are perturbed by more than one treatment.
Walker Wharton - One of the best experts on this subject based on the ideXlab platform.
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Targeted RNA Fingerprinting: the cloning of differentially-expressed cDNA fragments enriched for members of the zinc finger gene family.
Nucleic acids research, 1994Co-Authors: Brad Stone, Walker WhartonAbstract:We have developed and applied a modification of an 'RNA Fingerprinting' protocol previously published by Welsh and McClelland (Nucleic Acids Research 19: 5275-5279 1991) such that cDNA fragments which are both differentially-expressed and enriched for members of a specific gene family can readily be identified. cDNA fragments were amplified with an arbitrary primer initially used in the reverse transcription reaction in combination with a member of a primer set which corresponded to a conserved region within a specific gene family. This technique was used to isolate cDNAs encoding a recently described protein kinase as well as an unknown gene that contained a zinc finger. Several other known genes that contained a zinc finger domain and that were differentially-expressed were also isolated.
