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Papers by Robert Zagursky
Journal of Bacteriology, 2001
The advent of transcription profiling technologies has provided researchers with an unprecedented... more The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and agr , sarA , and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an agr - and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and p...
Gene, 1986
We describe a cloning-expression vector system for selecting DNA fragments containing open readin... more We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.
Expert Review of Vaccines
Gene Analysis Techniques, 1985
We have developed a rapid method for sequencing supercoiled plasmid DNA and bacteriophage k DNA. ... more We have developed a rapid method for sequencing supercoiled plasmid DNA and bacteriophage k DNA. The plasmid DNA is obtained from a 5-ml overnight culture using a rapid alkaline extraction method. The double-stranded lambda DNA is extracted from 0.75 ml of a standard liquid phage lysate using a modification of the diethylaminoethyl cellulose method. A synthetic oligonucleotide primer (12-18 bases) is annealed to the DNA, and the DNA is rapidly sequenced using the Sanger dideoxy chain-termination technique and reverse transcriptase. The advantages of these methods are 1) no cloning of the DNA is necessary, 2) one can rapidly and easily extract DNA, sequence it, and obtain the sequence in one day, and 3) both strands of the DNA can be sequenced from one molecule.
Science, 1987
A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleoti... more A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different chemically tuned succinylfluorescein dye distinguished by its fluorescent emission is described. Avian myeloblastosis virus reverse transcriptase is used in a modified dideoxy DNA sequencing protocol to produce a complete set of fluorescence-tagged fragments in one reaction mixture. These DNA fragments are resolved by polyacrylamide gel electrophoresis in one sequencing lane and are identified by a fluorescence detection system specifically matched to the emission characteristics of this dye set. A scanning system allows multiple samples to be run simultaneously and computer-based automatic base sequence identifications to be made. The sequence analysis of M13 phage DNA made with this system is described.
Developments in biologicals, 2000
Currently, there is an extensive and unprecedented effort to obtain the complete nucleotide seque... more Currently, there is an extensive and unprecedented effort to obtain the complete nucleotide sequence of the complex genomes of many micro-organisms. In this post-genomic era, based on the availability of the entire genome sequence of an organism, three new disciplines of molecular biology have emerged: genomics, transcriptional profiling and proteomics. All these technologies have the potential to accelerate the process of identifying protective protein antigens as subunit vaccine targets as well as validating and extending the range of available candidate antigens. The progress of these technologies has led to the origination of the science of bioinformatics for management and critical evaluation of the large amount of information generated. Although genomics, transcriptional profiling and proteomics are each based on different principles, there is considerable synergy between them. Appropriate application of any one, or a combination of two or more of these approaches, coupled wit...
The bacteriophage lambda genes exo and bet, whose products (lambda exonuclease and beta protein, ... more The bacteriophage lambda genes exo and bet, whose products (lambda exonuclease and beta protein, respectively; Red phenotype) mediate homologous recombination of lambda phages, have been cloned under lacPO lacIq control on multi-copy plasmids. Induction of recA3 cells harboring these plasmids with isopropylthiogalactoside (IPTG) resulted in lambda exonuclease levels (assayed in vitro) that were proportional to the time of induction (for at least 4 h); recombination of lambda Red- phages in vivo was similarly inducible. Only one out of 25 bet delta plasmids (constructed by a variety of in vitro techniques) expressed lambda exonuclease, a result consistent with the polarity of several known phage bet mutations. A general method for transferring phage exo and bet mutations to plasmids was devised and plasmids bearing polar (bet3) and nonpolar (bet113) mutations were constructed. Mutant derivatives of the plasmid showed the same complementation pattern as analogous phage red mutants. Wh...
INSTRUCTIONS Credit can now be obtained, free for a limited time, by reading the review article i... more INSTRUCTIONS Credit can now be obtained, free for a limited time, by reading the review article in this issue and completing all activity components. Please note the instructions listed below: Review the target audience, learning objectives and all disclosures. Complete the pre-test online at http://www.annallergy.org (click on the CME heading). Follow the online instructions to read the full version of the article; reflect on all content as to how it may be applicable to your practice. Complete the post-test/evaluation and claim credit earned; at this time, you will have earned up to 1.0 AMA PRA Category 1 Credit TM. Please note that the minimum passing score on the post-test is 70%.
The Journal of Allergy and Clinical Immunology: In Practice
Infection and Immunity
should read as follows. ".. . Oligonucleotides used for the amplification of these 2086 genes wer... more should read as follows. ".. . Oligonucleotides used for the amplification of these 2086 genes were 5LIP2996 and N2996SPH. The same oligonucleotide PCR primers were used for all five strains. The restriction site BamHI was. .. ." Page 2091, Table 2: Row 18 labeled "5LIP771" should not be included in the table.
Journal of Bacteriology, 2001
The advent of transcription profiling technologies has provided researchers with an unprecedented... more The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and agr , sarA , and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an agr - and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and p...
Gene, 1986
We describe a cloning-expression vector system for selecting DNA fragments containing open readin... more We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.
Expert Review of Vaccines
Gene Analysis Techniques, 1985
We have developed a rapid method for sequencing supercoiled plasmid DNA and bacteriophage k DNA. ... more We have developed a rapid method for sequencing supercoiled plasmid DNA and bacteriophage k DNA. The plasmid DNA is obtained from a 5-ml overnight culture using a rapid alkaline extraction method. The double-stranded lambda DNA is extracted from 0.75 ml of a standard liquid phage lysate using a modification of the diethylaminoethyl cellulose method. A synthetic oligonucleotide primer (12-18 bases) is annealed to the DNA, and the DNA is rapidly sequenced using the Sanger dideoxy chain-termination technique and reverse transcriptase. The advantages of these methods are 1) no cloning of the DNA is necessary, 2) one can rapidly and easily extract DNA, sequence it, and obtain the sequence in one day, and 3) both strands of the DNA can be sequenced from one molecule.
Science, 1987
A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleoti... more A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different chemically tuned succinylfluorescein dye distinguished by its fluorescent emission is described. Avian myeloblastosis virus reverse transcriptase is used in a modified dideoxy DNA sequencing protocol to produce a complete set of fluorescence-tagged fragments in one reaction mixture. These DNA fragments are resolved by polyacrylamide gel electrophoresis in one sequencing lane and are identified by a fluorescence detection system specifically matched to the emission characteristics of this dye set. A scanning system allows multiple samples to be run simultaneously and computer-based automatic base sequence identifications to be made. The sequence analysis of M13 phage DNA made with this system is described.
Developments in biologicals, 2000
Currently, there is an extensive and unprecedented effort to obtain the complete nucleotide seque... more Currently, there is an extensive and unprecedented effort to obtain the complete nucleotide sequence of the complex genomes of many micro-organisms. In this post-genomic era, based on the availability of the entire genome sequence of an organism, three new disciplines of molecular biology have emerged: genomics, transcriptional profiling and proteomics. All these technologies have the potential to accelerate the process of identifying protective protein antigens as subunit vaccine targets as well as validating and extending the range of available candidate antigens. The progress of these technologies has led to the origination of the science of bioinformatics for management and critical evaluation of the large amount of information generated. Although genomics, transcriptional profiling and proteomics are each based on different principles, there is considerable synergy between them. Appropriate application of any one, or a combination of two or more of these approaches, coupled wit...
The bacteriophage lambda genes exo and bet, whose products (lambda exonuclease and beta protein, ... more The bacteriophage lambda genes exo and bet, whose products (lambda exonuclease and beta protein, respectively; Red phenotype) mediate homologous recombination of lambda phages, have been cloned under lacPO lacIq control on multi-copy plasmids. Induction of recA3 cells harboring these plasmids with isopropylthiogalactoside (IPTG) resulted in lambda exonuclease levels (assayed in vitro) that were proportional to the time of induction (for at least 4 h); recombination of lambda Red- phages in vivo was similarly inducible. Only one out of 25 bet delta plasmids (constructed by a variety of in vitro techniques) expressed lambda exonuclease, a result consistent with the polarity of several known phage bet mutations. A general method for transferring phage exo and bet mutations to plasmids was devised and plasmids bearing polar (bet3) and nonpolar (bet113) mutations were constructed. Mutant derivatives of the plasmid showed the same complementation pattern as analogous phage red mutants. Wh...
INSTRUCTIONS Credit can now be obtained, free for a limited time, by reading the review article i... more INSTRUCTIONS Credit can now be obtained, free for a limited time, by reading the review article in this issue and completing all activity components. Please note the instructions listed below: Review the target audience, learning objectives and all disclosures. Complete the pre-test online at http://www.annallergy.org (click on the CME heading). Follow the online instructions to read the full version of the article; reflect on all content as to how it may be applicable to your practice. Complete the post-test/evaluation and claim credit earned; at this time, you will have earned up to 1.0 AMA PRA Category 1 Credit TM. Please note that the minimum passing score on the post-test is 70%.
The Journal of Allergy and Clinical Immunology: In Practice
Infection and Immunity
should read as follows. ".. . Oligonucleotides used for the amplification of these 2086 genes wer... more should read as follows. ".. . Oligonucleotides used for the amplification of these 2086 genes were 5LIP2996 and N2996SPH. The same oligonucleotide PCR primers were used for all five strains. The restriction site BamHI was. .. ." Page 2091, Table 2: Row 18 labeled "5LIP771" should not be included in the table.