Charles Nicolet - Academia.edu (original) (raw)

Papers by Charles Nicolet

Journal of Immunology, Dec 15, 1991

Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive... more Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive molecules on human mononuclear cells.

Journal of Immunology, Nov 15, 1995

Promoter analysis of an interferon-inducible gene associated with macrophage activation.

Journal of biomolecular techniques, Sep 1, 2010

CF-34 The UC Davis Genome Center (GC) opened several years ago in a state-of-the-art facility and... more CF-34 The UC Davis Genome Center (GC) opened several years ago in a state-of-the-art facility and combines bench science with computational approaches to investigate questions in genomics. An important part of the GC is its five technology service cores providing access to enabling technologies to researchers within and outside the UC Davis campus. The DNA Technologies and Expression Analysis Cores are linked entities providing numerous services related to genotyping, genome structure, and sequencing. We operate three Illumina Genome Analyzer high throughput sequencers and two paired end modules. These instruments have been involved in numerous projects, including ChIP-seq, mRNA-seq, de novo genome assembly, and resequencing for SNP discovery. The Bioinformatics Core, another service entity associated with the GC, has been invaluable in rapidly allowing this instrumentation to provide full research value to the UC Davis campus. The DNA Technologies Core also utilizes all the available Illumina platforms for SNP genotyping, including the Golden Gate assay on both the iScan and BeadXpress, and the Infinium assay with associated Tecan liquid handling capability. The Expression Analysis Core provides array services and analysis on a subset of the available commercial platforms, focusing our efforts on Illumina expression arrays, as well as on NimbleGen and Agilent custom arrays. Because of local scientific expertise provided in the GC there is an emphasis on services related to chromatin immunoprecipitation (ChIP) techniques, a methodology generally unavailable in most technology service cores. We offer hands-on workshops in the basic manipulations required for ChIP as well as carrying out ChIP as a service with user-provided cells and antibodies. More information is available at http://genomecenter.ucdavis.edu/corefacilities.html.

Theoretical and Applied Genetics, May 18, 2009

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapp... more Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR ampliWcation. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very eYcient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach. Communicated by A. Schulman.

Cancer Research, 2018

DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, lat... more DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, late-replicating regions termed Partially Methylated Domains (PMDs). We profiled 39 diverse primary tumors and 8 matched adjacent tissues using Whole-Genome Bisulfite Sequencing (WGBS), and analyzed them alongside 343 additional human and 206 mouse WGBS datasets. We identified a local CpG sequence context associated with preferential hypomethylation in PMDs. Analysis of CpGs in this context (“Solo-WCGWs”) revealed previously undetected PMD hypomethylation in almost all healthy tissue types. PMD hypomethylation increases with age in both fetal and postnatal tissues, appearing to track accumulation of cell divisions beginning early in life. In cancer, the degree of PMD hypomethylation correlates with somatic mutation density and expression of cell-cycle dependent genes, reinforcing its mitotic clock-like role. We propose that late replication leads to progressive methylation loss throughout a...

Very little is known about the molecular mechanism of nucleotide excision repair in eukaryotes. S... more Very little is known about the molecular mechanism of nucleotide excision repair in eukaryotes. Studies on human cells have been stimulated by the availability of excision repair-defective cell lines from patients suffering from the autosomal recessive disease xeroderma pigmentosum (XP). Such studies have contributed significantly to an understanding of the genetic complexity of excision repair in human cells. However, to date, no human excision repair genes or gene products known to complement the repair defect in XP cells have been isolated. The yeast Saccharomyces cerevisiae is an interesting model for exploring the molecular mechanism of nucleotide excision repair in eukaryotic cells. As is true in human cells, multiple yeast genes are involved and at least five genes are required for the specific incision of UV-irradiated DNA in vivo. These five genes have been isolated by molecular cloning and the nucleotide sequences of four of them have been determined. Each of these cloned genes is being used for overexpression of protein.

As technology changes, the ability of core facilities to address the needs of their customers is ... more As technology changes, the ability of core facilities to address the needs of their customers is critical to maintaining relevance in a competitive field. The Genomic Variation Research Group (GVRG), in conjunction with the DNA Sequencing (DSRG) and Nucleic Acids (NARG) research groups, has put together a comprehensive survey of the types of genomic technologies and their applications currently in place at core facilities. This survey will also serve as a guide for other genomic research groups on how to best serve the needs of the ABRF membership. The approximately 10-minute survey was sent out to the ABRF membership as well as the discussion group forum for respondents. The questions ranged from sample types utilized to usage percentages of certain applications including the latest sequencing platforms. As these new sequencing technologies have changed the landscape of tools available to researchers we sought to understand the impact on the platforms previously employed. The surve...

Next generation sequencing (NGS) has dramatically expanded the potential for novel genomics disco... more Next generation sequencing (NGS) has dramatically expanded the potential for novel genomics discoveries, but the wide variety of platforms, protocols, and performance has created the need for reference data sets to understand the sources of variation in results. The goals of the ABRF-NGS Study are to use standard references to evaluate the performance of NGS platforms and to identify optimal methods and best practices. For the first phase of this study, over 20 core facility laboratories performed replicate RNA-seq experiments, using titrated reference RNA standards and a set of synthetic RNA spike-ins, evaluated over a wide range of methods: polyA-enriched, ribo-depleted, size-specific fractionations, and degraded RNA, on six NGS platforms (Illumina HiSeq 2000/2500 and MiSeq, Life Technologies PGM and Proton, Roche 454 GS FLX+, and PacBio RS). Two RT-qPCR data sets were used as orthogonal tools to gauge the RNA-seq results. The results show high intra-platform consistency and inter-platform concordance for expression measures, but also demonstrate highly variable rates of efficiency and costs for splice isoform detection between platforms. The data also add evidence that ribosomal RNA depletion can both salvage degraded RNA samples and be readily compared to polyA-enriched fractions. Comparisons of alternative aligners for each platform show that algorithm choice affects mapping rates and transcript coverage more than gene quantification. Surrogate variable analysis (SVA) proved to be an optimal method to combine data within and between platforms, increasing sensitivity and reducing false positives by over 90%. Taken together, these data represent a broad cross-platform characterization of RNA standards and provide a comprehensive comparison of results from degraded, full-length, and size-selected RNA across the latest NGS platforms. The next phase of this study is focusing on use of DNA reference standards. Results of the ABRF-NGS Study provide a broad foundation for cross-platform standardization, evaluation, and improvement of NGS applications.

Journal of biomolecular techniques, 2012

•To query the ABRF membership and scientists at-large concerning the current state of funding in ... more •To query the ABRF membership and scientists at-large concerning the current state of funding in service-oriented laboratories. •Questions were designed to elicit responses concerning service offerings, cost recovery, capital equipment funding, and future outlook. Objectives Materials and Methods •The survey was web-based utilizing the on-line survey software “SurveyMonkey”

© The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Sin... more © The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR ampliWcation. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1 % for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays wer...

Tumor Biology, 1997

Aberrant glycosylation of mucins on the surface of adenocarcinomas leads to exposure of novel tum... more Aberrant glycosylation of mucins on the surface of adenocarcinomas leads to exposure of novel tumor-associated epitopes potentially recognizable by the immune system. Monoclonal antibodies (mAbs) have been developed against some of these epitopes. One such mAb, denoted CC49, recognizes the tumor-associated glycoprotein TAG-72. Most adenocarcinomas, including breast, colon, ovarian, prostate, and gastric, express some form of this molecule, recognizable by the CC49 antibody. The widespread distribution of the antigen on transformed cells makes the CC49 mAb a potentially powerful tool in numerous immunotherapy contexts. In the course of our studies with CC49 and certain of its molecularly engineered derivatives, we screened a number of human hematopoietic cell lines for TAG-72 expression by flow cytometry using CC49. We found that the T-cell line, Jurkat, had a higher level of CC49 mAb binding than any of the carcinoma cell lines previously evaluated in our laboratory. In addition, the myelomonocytic cell line Tf-1 and the erythroleukemia cell line K562 were also positive for CC49 mAb binding by flow-cytometric analysis. However, peripheral blood lymphocytes and certain other hematopoietic cell lines were not able to bind the CC49 mAb. Immunoblot analyses of cell extracts from the CC49 reactive lines indicated distinct protein species reactive with the CC49 antibody. In some instances, cells expressing these reactive proteins were susceptible to antibody-dependent cellular cytotoxicity using a chimeric derivative of the CC49 antibody. These results indicate that the cell membrane expression of molecules recognized by CC49 extends beyond adenocarcinomas to certain cell lines of hematopoietic origin.

The Journal of Immunology

We have investigated the regulation of an activation-associated guanylate-binding protein gene (m... more We have investigated the regulation of an activation-associated guanylate-binding protein gene (mGBP-1/mag-1) in murine macrophage cell lines in response to the cytokine IFN-gamma. One of the cell lines utilized (RAW 264.7) acquires the ability to kill tumor cells after IFN-gamma and LPS treatment, whereas the other (WEHI-3) does not. We previously have demonstrated that mGBP-1 is induced by IFN-gamma in RAW 264.7 but not WEHI-3 cells. Here we present information concerning the cloning, sequencing, and initial characterization of the upstream region of the mGBP-1 gene as a first step towards understanding the differential control of this gene in RAW 264.7 versus WEHI-3 cells. Genomic fragments encompassing a portion of the mGBP-1 5' flanking region were inserted into vectors containing a luciferase reporter gene. 928 bp of upstream sequence were found to be sufficient for IFN-gamma-mediated induction of luciferase activity in the RAW 264.7 cell line. Furthermore, sequences withi...

The Journal of Immunology

ABSTRACT

Nucleic Acids Research, 2009

Next-generation sequencing is revolutionizing the identification of transcription factor binding ... more Next-generation sequencing is revolutionizing the identification of transcription factor binding sites throughout the human genome. However, the bioinformatics analysis of large datasets collected using chromatin immunoprecipitation and highthroughput sequencing is often a roadblock that impedes researchers in their attempts to gain biological insights from their experiments. We have developed integrated peak-calling and analysis software (Sole-Search) which is available through a user-friendly interface and (i) converts raw data into a format for visualization on a genome browser, (ii) outputs ranked peak locations using a statistically based method that overcomes the significant problem of false positives, (iii) identifies the gene nearest to each peak, (iv) classifies the location of each peak relative to gene structure, (v) provides information such as the number of binding sites per chromosome and per gene and (vi) allows the user to determine overlap between two different experiments. In addition, the program performs an analysis of amplified and deleted regions of the input genome. This software is web-based and automated, allowing easy and immediate access to all investigators. We demonstrate the utility of our software by collecting, analyzing and comparing ChIP-seq data for six different human transcription factors/cell line combinations.

Transcription Factor Protocols

ABSTRACT

Multiple recent publications on RNA-Seq have demonstrated the power of next generation sequencing... more Multiple recent publications on RNA-Seq have demonstrated the power of next generation sequencing technologies in whole transcriptome analysis. The vendor specific protocols used for RNA library construction typically require at least 100ng of total RNA. However, under certain conditions such as single cells, stem cells, difficult to isolate cell types, or fractionated cancer cells, only a small amount of material is available. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several RNA amplification kits are available for amplification prior to library construction and next generation sequencing but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-Seq for picogram amounts of RNA. This study conducted by the DNA Sequencing Research Group (DSRG) focuses on the evaluation of amplification kits for RNA-Seq. Four commercial amplification kits were chosen: Ovation v2 (NuGEN Technologies), ...

Journal of biomolecular techniques, 2012

DNA methylation is an important epigenetic mechanism for regulating the activity of the genome. I... more DNA methylation is an important epigenetic mechanism for regulating the activity of the genome. Inter-individual differences in the epigenome, including the DNA methylome, are thought to account for the missing variance in disease susceptibility that has not been identified in Genome-Wide Association Studies (GWAS). Large-scale profiling of DNA methylation in population cohorts at the sample size of thousands to tens of thousands is necessary to characterize the epigenetic component of diseases susceptibility. Although whole genome bisulfite sequencing has been demonstrated in mammalian-size genomes, it is still too costly for a large sample size. In addition, only a very small fraction of CpG sites in the human genome is variable and carries information related to the epigenetic state of the cells, whereas the majority of CpG sites are static. For large-scale methylation sequencing projects, ideally the sequencing cost should be spent only on the informative sites in the genome. We...

Nature Biotechnology, 2021

In the version of this Article initially published, Extended Data Fig. 4 had incorrect coloration... more In the version of this Article initially published, Extended Data Fig. 4 had incorrect coloration and an incorrect legend. PacBio long reads matched against CLINVAR variants were incorrectly colored in light green instead of brown (top right of panel a). In the bottom shared legend (below panel c), Nanopore was incorrectly identified with a brown circle instead of dark green. This figure has been corrected with a new legend for CLINVAR/OMIM variant analysis (below panel b) and a separate, corrected legend for exam variant analysis at the bottom of the figure. The online version of the article has been updated.

Nature Biotechnology, 2021

Peer review information Nature Biotechnology thanks the anonymous reviewers for their contributio... more Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

Journal of Immunology, Dec 15, 1991

Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive... more Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive molecules on human mononuclear cells.

Journal of Immunology, Nov 15, 1995

Promoter analysis of an interferon-inducible gene associated with macrophage activation.

Journal of biomolecular techniques, Sep 1, 2010

CF-34 The UC Davis Genome Center (GC) opened several years ago in a state-of-the-art facility and... more CF-34 The UC Davis Genome Center (GC) opened several years ago in a state-of-the-art facility and combines bench science with computational approaches to investigate questions in genomics. An important part of the GC is its five technology service cores providing access to enabling technologies to researchers within and outside the UC Davis campus. The DNA Technologies and Expression Analysis Cores are linked entities providing numerous services related to genotyping, genome structure, and sequencing. We operate three Illumina Genome Analyzer high throughput sequencers and two paired end modules. These instruments have been involved in numerous projects, including ChIP-seq, mRNA-seq, de novo genome assembly, and resequencing for SNP discovery. The Bioinformatics Core, another service entity associated with the GC, has been invaluable in rapidly allowing this instrumentation to provide full research value to the UC Davis campus. The DNA Technologies Core also utilizes all the available Illumina platforms for SNP genotyping, including the Golden Gate assay on both the iScan and BeadXpress, and the Infinium assay with associated Tecan liquid handling capability. The Expression Analysis Core provides array services and analysis on a subset of the available commercial platforms, focusing our efforts on Illumina expression arrays, as well as on NimbleGen and Agilent custom arrays. Because of local scientific expertise provided in the GC there is an emphasis on services related to chromatin immunoprecipitation (ChIP) techniques, a methodology generally unavailable in most technology service cores. We offer hands-on workshops in the basic manipulations required for ChIP as well as carrying out ChIP as a service with user-provided cells and antibodies. More information is available at http://genomecenter.ucdavis.edu/corefacilities.html.

Theoretical and Applied Genetics, May 18, 2009

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapp... more Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR ampliWcation. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very eYcient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach. Communicated by A. Schulman.

Cancer Research, 2018

DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, lat... more DNA methylation loss occurs frequently in cancer genomes, primarily within lamina-associated, late-replicating regions termed Partially Methylated Domains (PMDs). We profiled 39 diverse primary tumors and 8 matched adjacent tissues using Whole-Genome Bisulfite Sequencing (WGBS), and analyzed them alongside 343 additional human and 206 mouse WGBS datasets. We identified a local CpG sequence context associated with preferential hypomethylation in PMDs. Analysis of CpGs in this context (“Solo-WCGWs”) revealed previously undetected PMD hypomethylation in almost all healthy tissue types. PMD hypomethylation increases with age in both fetal and postnatal tissues, appearing to track accumulation of cell divisions beginning early in life. In cancer, the degree of PMD hypomethylation correlates with somatic mutation density and expression of cell-cycle dependent genes, reinforcing its mitotic clock-like role. We propose that late replication leads to progressive methylation loss throughout a...

Very little is known about the molecular mechanism of nucleotide excision repair in eukaryotes. S... more Very little is known about the molecular mechanism of nucleotide excision repair in eukaryotes. Studies on human cells have been stimulated by the availability of excision repair-defective cell lines from patients suffering from the autosomal recessive disease xeroderma pigmentosum (XP). Such studies have contributed significantly to an understanding of the genetic complexity of excision repair in human cells. However, to date, no human excision repair genes or gene products known to complement the repair defect in XP cells have been isolated. The yeast Saccharomyces cerevisiae is an interesting model for exploring the molecular mechanism of nucleotide excision repair in eukaryotic cells. As is true in human cells, multiple yeast genes are involved and at least five genes are required for the specific incision of UV-irradiated DNA in vivo. These five genes have been isolated by molecular cloning and the nucleotide sequences of four of them have been determined. Each of these cloned genes is being used for overexpression of protein.

As technology changes, the ability of core facilities to address the needs of their customers is ... more As technology changes, the ability of core facilities to address the needs of their customers is critical to maintaining relevance in a competitive field. The Genomic Variation Research Group (GVRG), in conjunction with the DNA Sequencing (DSRG) and Nucleic Acids (NARG) research groups, has put together a comprehensive survey of the types of genomic technologies and their applications currently in place at core facilities. This survey will also serve as a guide for other genomic research groups on how to best serve the needs of the ABRF membership. The approximately 10-minute survey was sent out to the ABRF membership as well as the discussion group forum for respondents. The questions ranged from sample types utilized to usage percentages of certain applications including the latest sequencing platforms. As these new sequencing technologies have changed the landscape of tools available to researchers we sought to understand the impact on the platforms previously employed. The surve...

Next generation sequencing (NGS) has dramatically expanded the potential for novel genomics disco... more Next generation sequencing (NGS) has dramatically expanded the potential for novel genomics discoveries, but the wide variety of platforms, protocols, and performance has created the need for reference data sets to understand the sources of variation in results. The goals of the ABRF-NGS Study are to use standard references to evaluate the performance of NGS platforms and to identify optimal methods and best practices. For the first phase of this study, over 20 core facility laboratories performed replicate RNA-seq experiments, using titrated reference RNA standards and a set of synthetic RNA spike-ins, evaluated over a wide range of methods: polyA-enriched, ribo-depleted, size-specific fractionations, and degraded RNA, on six NGS platforms (Illumina HiSeq 2000/2500 and MiSeq, Life Technologies PGM and Proton, Roche 454 GS FLX+, and PacBio RS). Two RT-qPCR data sets were used as orthogonal tools to gauge the RNA-seq results. The results show high intra-platform consistency and inter-platform concordance for expression measures, but also demonstrate highly variable rates of efficiency and costs for splice isoform detection between platforms. The data also add evidence that ribosomal RNA depletion can both salvage degraded RNA samples and be readily compared to polyA-enriched fractions. Comparisons of alternative aligners for each platform show that algorithm choice affects mapping rates and transcript coverage more than gene quantification. Surrogate variable analysis (SVA) proved to be an optimal method to combine data within and between platforms, increasing sensitivity and reducing false positives by over 90%. Taken together, these data represent a broad cross-platform characterization of RNA standards and provide a comprehensive comparison of results from degraded, full-length, and size-selected RNA across the latest NGS platforms. The next phase of this study is focusing on use of DNA reference standards. Results of the ABRF-NGS Study provide a broad foundation for cross-platform standardization, evaluation, and improvement of NGS applications.

Journal of biomolecular techniques, 2012

•To query the ABRF membership and scientists at-large concerning the current state of funding in ... more •To query the ABRF membership and scientists at-large concerning the current state of funding in service-oriented laboratories. •Questions were designed to elicit responses concerning service offerings, cost recovery, capital equipment funding, and future outlook. Objectives Materials and Methods •The survey was web-based utilizing the on-line survey software “SurveyMonkey”

© The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Sin... more © The Author(s) 2009. This article is published with open access at Springerlink.com Abstract Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR ampliWcation. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1 % for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays wer...

Tumor Biology, 1997

Aberrant glycosylation of mucins on the surface of adenocarcinomas leads to exposure of novel tum... more Aberrant glycosylation of mucins on the surface of adenocarcinomas leads to exposure of novel tumor-associated epitopes potentially recognizable by the immune system. Monoclonal antibodies (mAbs) have been developed against some of these epitopes. One such mAb, denoted CC49, recognizes the tumor-associated glycoprotein TAG-72. Most adenocarcinomas, including breast, colon, ovarian, prostate, and gastric, express some form of this molecule, recognizable by the CC49 antibody. The widespread distribution of the antigen on transformed cells makes the CC49 mAb a potentially powerful tool in numerous immunotherapy contexts. In the course of our studies with CC49 and certain of its molecularly engineered derivatives, we screened a number of human hematopoietic cell lines for TAG-72 expression by flow cytometry using CC49. We found that the T-cell line, Jurkat, had a higher level of CC49 mAb binding than any of the carcinoma cell lines previously evaluated in our laboratory. In addition, the myelomonocytic cell line Tf-1 and the erythroleukemia cell line K562 were also positive for CC49 mAb binding by flow-cytometric analysis. However, peripheral blood lymphocytes and certain other hematopoietic cell lines were not able to bind the CC49 mAb. Immunoblot analyses of cell extracts from the CC49 reactive lines indicated distinct protein species reactive with the CC49 antibody. In some instances, cells expressing these reactive proteins were susceptible to antibody-dependent cellular cytotoxicity using a chimeric derivative of the CC49 antibody. These results indicate that the cell membrane expression of molecules recognized by CC49 extends beyond adenocarcinomas to certain cell lines of hematopoietic origin.

The Journal of Immunology

We have investigated the regulation of an activation-associated guanylate-binding protein gene (m... more We have investigated the regulation of an activation-associated guanylate-binding protein gene (mGBP-1/mag-1) in murine macrophage cell lines in response to the cytokine IFN-gamma. One of the cell lines utilized (RAW 264.7) acquires the ability to kill tumor cells after IFN-gamma and LPS treatment, whereas the other (WEHI-3) does not. We previously have demonstrated that mGBP-1 is induced by IFN-gamma in RAW 264.7 but not WEHI-3 cells. Here we present information concerning the cloning, sequencing, and initial characterization of the upstream region of the mGBP-1 gene as a first step towards understanding the differential control of this gene in RAW 264.7 versus WEHI-3 cells. Genomic fragments encompassing a portion of the mGBP-1 5' flanking region were inserted into vectors containing a luciferase reporter gene. 928 bp of upstream sequence were found to be sufficient for IFN-gamma-mediated induction of luciferase activity in the RAW 264.7 cell line. Furthermore, sequences withi...

The Journal of Immunology

ABSTRACT

Nucleic Acids Research, 2009

Next-generation sequencing is revolutionizing the identification of transcription factor binding ... more Next-generation sequencing is revolutionizing the identification of transcription factor binding sites throughout the human genome. However, the bioinformatics analysis of large datasets collected using chromatin immunoprecipitation and highthroughput sequencing is often a roadblock that impedes researchers in their attempts to gain biological insights from their experiments. We have developed integrated peak-calling and analysis software (Sole-Search) which is available through a user-friendly interface and (i) converts raw data into a format for visualization on a genome browser, (ii) outputs ranked peak locations using a statistically based method that overcomes the significant problem of false positives, (iii) identifies the gene nearest to each peak, (iv) classifies the location of each peak relative to gene structure, (v) provides information such as the number of binding sites per chromosome and per gene and (vi) allows the user to determine overlap between two different experiments. In addition, the program performs an analysis of amplified and deleted regions of the input genome. This software is web-based and automated, allowing easy and immediate access to all investigators. We demonstrate the utility of our software by collecting, analyzing and comparing ChIP-seq data for six different human transcription factors/cell line combinations.

Transcription Factor Protocols

ABSTRACT

Multiple recent publications on RNA-Seq have demonstrated the power of next generation sequencing... more Multiple recent publications on RNA-Seq have demonstrated the power of next generation sequencing technologies in whole transcriptome analysis. The vendor specific protocols used for RNA library construction typically require at least 100ng of total RNA. However, under certain conditions such as single cells, stem cells, difficult to isolate cell types, or fractionated cancer cells, only a small amount of material is available. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several RNA amplification kits are available for amplification prior to library construction and next generation sequencing but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-Seq for picogram amounts of RNA. This study conducted by the DNA Sequencing Research Group (DSRG) focuses on the evaluation of amplification kits for RNA-Seq. Four commercial amplification kits were chosen: Ovation v2 (NuGEN Technologies), ...

Journal of biomolecular techniques, 2012

DNA methylation is an important epigenetic mechanism for regulating the activity of the genome. I... more DNA methylation is an important epigenetic mechanism for regulating the activity of the genome. Inter-individual differences in the epigenome, including the DNA methylome, are thought to account for the missing variance in disease susceptibility that has not been identified in Genome-Wide Association Studies (GWAS). Large-scale profiling of DNA methylation in population cohorts at the sample size of thousands to tens of thousands is necessary to characterize the epigenetic component of diseases susceptibility. Although whole genome bisulfite sequencing has been demonstrated in mammalian-size genomes, it is still too costly for a large sample size. In addition, only a very small fraction of CpG sites in the human genome is variable and carries information related to the epigenetic state of the cells, whereas the majority of CpG sites are static. For large-scale methylation sequencing projects, ideally the sequencing cost should be spent only on the informative sites in the genome. We...

Nature Biotechnology, 2021

In the version of this Article initially published, Extended Data Fig. 4 had incorrect coloration... more In the version of this Article initially published, Extended Data Fig. 4 had incorrect coloration and an incorrect legend. PacBio long reads matched against CLINVAR variants were incorrectly colored in light green instead of brown (top right of panel a). In the bottom shared legend (below panel c), Nanopore was incorrectly identified with a brown circle instead of dark green. This figure has been corrected with a new legend for CLINVAR/OMIM variant analysis (below panel b) and a separate, corrected legend for exam variant analysis at the bottom of the figure. The online version of the article has been updated.

Nature Biotechnology, 2021

Peer review information Nature Biotechnology thanks the anonymous reviewers for their contributio... more Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.