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D H Gelfand

Publications and source records attributed to D H Gelfand.

At least 19 recordsLinked to original sources
PubMed2000

Transcript quantitation in total yeast cellular RNA using kinetic PCR.

Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT-PCR, kRT-PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT-PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of +/-20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators.

DNA Primers↗
PubMed1996

Selective amplification of RNA utilizing the nucleotide analog dITP and Thermus thermophilus DNA polymerase.

The ability to selectively amplify RNA in the presence of genomic DNA of analogous sequence is cumbersome and requires implementation of critical controls for genes lacking introns. The convenient approaches of either designing oligonucleotide primers at the splice junction or differentiating the target sequence based on the size difference obtained by the presence of the intron are not possible. Our strategy for the selective amplification of RNA targets is based on the enzymology of a single thermostable DNA polymerase and the ability to modulate the strand separation temperature requirements for PCR amplification. Following reverse transcription of the RNA by recombinant Thermus thermophilus DNA polymerase (rTth pol), the resulting RNAxDNA hybrid is digested by the RNase H activity of rTth pol, allowing the PCR primer to hybridize and initiate second-strand cDNA synthesis. Substitution of one or more conventional nucleotides with nucleotide analogs that decrease base stacking interactions and/or hydrogen bonding (e.g. hydroxymethyldUTP or dITP) during the first- and second-strand cDNA synthesis step reduces the strand separation temperature of the resultant DNAxDNA duplex. Alteration of the thermal cycling parameters of the subsequent PCR amplification, such that the strand separation temperature is below that required for denaturation of genomic duplex DNA composed of standard nucleotides, prevents the genomic DNA from being denatured and therefore amplified.

DNA-Directed DNA Polymerase↗
PubMed1994

Cloning, sequencing, and expression of RecA proteins from three distantly related thermophilic eubacteria.

Sequences of the recA genes of the highly divergent thermopholic eubacteria Thermus aquaticus (and Thermus thermophilus), Thermotoga maritima, and Aquifex pyrophilus were determined from fragments derived by polymerase chain reaction (PCR) with degenerate primers and from inverse PCR products obtained using unique primers based on the fragment sequences. The source of the PCR products was verified by Southern hybridization. Complete PCR-derived recA genes were cloned into an expression vector regulated by a temperature-sensitive lambda-repressor, and independently derived clones expressing thermostable recA were selected. DNA sequences were verified to be authentic by direct cycle-sequencing of PCR products and/or sequencing of several clones. In contrast to Escherichia coli RecA protein, all the purified thermophilic RecA proteins exhibited single-stranded DNA-dependent ATPase activity optima above 70 degrees C. Phylogenetic analysis of RecA sequences suggested that the thermophilic RecA proteins were at least as different from one another as were Gram-positive organisms, mesophilic Gram-negative organisms, and cyanobacteria. In spite of substantial sequence divergence, interesting characteristics of the thermostable RecA proteins included increased valine content, common amino acid replacements at two highly conserved sites, and an increase in the calculated isoelectric point of approximately a full pH unit.

Adenosine Triphosphate↗
PubMed1993

High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity.

The Thermus aquaticus DNA polymerase I (Taq Pol I) gene was cloned into a plasmid expression vector that utilizes the strong bacteriophage lambda PL promoter. A truncated form of Taq Pol I was also constructed. The two constructs made it possible to compare the full-length 832-amino-acid Taq Pol I and a deletion derivative encoding a 544-amino-acid translation product, the Stoffel fragment. Upon heat induction, the 832-amino-acid construct produced 1-2% of total protein as Taq Pol I. The induced 544-amino-acid construct produced 3% of total protein as Stoffel fragment. Enzyme purification included cell lysis, heat treatment followed by Polymin P precipitation of nucleic acids, phenyl sepharose column chromatography, and heparin-Sepharose column chromatography. For full-length 94-kD Taq Pol I, yield was 3.26 x 10(7) units of activity from 165 grams wet weight cell paste. For the 61-kD Taq Pol I Stoffel fragment, the yield was 1.03 x 10(6) units of activity from 15.6 grams wet weight cell paste. The two enzymes have maximal activity at 75 degrees C to 80 degrees C, 2-4 mM MgCl2 and 10-55 mM KCl. The nature of the substrate determines the precise conditions for maximal enzyme activity. For both proteins, MgCl2 is the preferred cofactor compared to MnCl2, CoCl2, and NiCl2. The full-length Taq Pol I has an activity half-life of 9 min at 97.5 degrees C. The Stoffel fragment has a half-life of 21 min at 97.5 degrees C. Taq Pol I contains a polymerization-dependent 5' to 3' exonuclease activity whereas the Stoffel fragment, deleted for the 5' to 3' exonuclease domain, does not possess that activity. A comparison is made among thermostable DNA polymerases that have been characterized; specific activities of 292,000 units/mg for Taq Pol I and 369,000 units/mg for the Stoffel fragment are the highest reported.

Amino Acid Sequence↗
PubMed1991

Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.

Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.

Amino Acid Sequence↗
PubMed1991

Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'----3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.

Base Sequence↗
PubMed1991

Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase.

A recombinant DNA polymerase derived from the thermophilic eubacterium Thermus thermophilus (Tth pol) was found to possess very efficient reverse transcriptase (RT) activity in the presence of MnCl2. Many of the problems typically associated with the high degree of secondary structure present in RNA are minimized by using a thermostable DNA polymerase for reverse transcription, and predominantly full-length products can be obtained. The cDNA can also be amplified in the polymerase chain reaction (PCR) with the same enzyme. The Tth pol was observed to be greater than 100-fold more efficient in a coupled RT/PCR than the analogous DNA polymerase from Thermus aquaticus (Taq pol). The sensitivity of the reactions performed by Tth pol allowed for the detection of ethidium bromide stained products starting with as little as 100 copies of synthetic cRNA. Similar results were also obtained with RNA from a Philadelphia-chromosome positive cell line. Detection of IL-1 alpha mRNA was possible starting with 80 pg of total cellular RNA. The ability of Tth pol to perform both reverse transcription and DNA amplification will undoubtedly prove useful in the detection, quantitation, and cloning of cellular and viral RNA.

Base Sequence↗
PubMed1990

Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.

Arabidopsis Proteins↗
PubMed1989

Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.

The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction method for amplifying DNA. We report the cloning and expression of Taq DNA polymerase in Escherichia coli. From a lambda gt11:Taq library we identified a Taq DNA fragment encoding an epitope of Taq DNA polymerase via antibody probing. The fusion protein from the lambda gt11:Taq candidate selected an antibody from an anti-Taq polymerase polyclonal antiserum which reacted with Taq polymerase on Western blots. We used the lambda gt11 clone to identify Taq polymerase clones from a lambda Ch35:Taq library. The complete Taq DNA polymerase gene has 2499 base pairs. From the predicted 832-amino acid sequence of the Taq DNA polymerase gene, Taq DNA polymerase has significant similarity to E. coli DNA polymerase I. We subcloned and expressed appropriate portions of the insert from a lambda Ch35 library candidate to yield thermostable, active, truncated, or full-length forms of the protein in E. coli under control of the lac promoter.

Amino Acid Sequence↗
PubMed1988

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

Cloning, Molecular↗
PubMed1988

DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2'-deoxyguanosine was used to sequence through G + C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.

Autoanalysis↗
PubMed1978

Isolation and characterization of ColE1-derived plasmid copy-number mutant.

The plasmid pBGP120 is a ColE1 derivative that contains elements of the Escherichia coli lac operon and the Tn3 transposon. We have selected and isolated a copy-number mutant of pBGP120. In exponentially growing cultures, the copy-number mutant, pOP1, represents approximately 30% of total intracellular DNA compared to about 5% for pBGP120. Plasmid-encoded beta-galactosidase monomer can represent 50% of newly synthesized protein in cells carrying pOP1. pOP1 is structurally unstable in certain genetic backgrounds and under certain growth conditions, breaking down to a smaller sized plasmid that retains the DNA overproducer phenotype and the Tn3 transposon. The smaller overproducer plasmid, pOP1delta6, is generated by a continuous deletion of sequences located between one end of the Tn3 transposon and a site about 630 nucleotides from the EcoRI site in the beta-galactosidase structural gene of pOP1. pOP1delta6 retains the ColE1 origin of replication but has lost the lac promotor and operator and most of the beta-galactosidase structural gene. pOP1delta6 exists at approximately 210 copies per chromosome in exponentially growing cells.

Bacteriocin Plasmids↗
PubMed1978

Regulated expression by readthrough translation from a plasmid-encoded beta-galactosidase.

We have characterized expression of beta-galactosidase from a plasmid cloning vehicle, pBGP120, which carries most of the lacZ gene and contains a single EcoRI site near the end of lacZ. In addition, we have examined expression of heterologous DNA inserted at the position of the EcoRI site. The EcoRI site was shown to be within the sequence coding for beta-galactosidase and its precise location and phase were deduced. Insertion of heterologous EcoRI-generated DNA fragments altered the molecular weight of the plasmid-encoded beta-galactosidase polypeptide. Those insertions that were in the correct phase were expressed at a high level as a fused protein. The different forms of beta-galactosidase polypeptides produced by various hybrid plasmids were all stable proteins. The level of expression of the plasmid-encoded beta-galactosidase was several times higher than maximal expression of chromosome-encoded beta-galactosidase, suggesting that expression is proportional to gene copy number. The expression of the plasmid lacZ gene was controlled by cyclic AMP. When grown in a cya strain (DG74), expression was dependent on exogenous cyclic AMP. Although in normal strains there was insufficient lac repressor to inactivate all copies of the plasmid, repressor regulation was restored when the plasmid was grown in a strain (M96) that overproduces the lac repressor.

Cyclic AMP↗
PubMed1977

Ribosomal RNA genes of Saccharomyces cerevisiae. I. Physical map of the repeating unit and location of the regions coding for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs.

The organization of the ribosomal DNA repeating unit from Saccharomyces cerevisiae has been analyzed. A cloned ribosomal DNA repeating unit has been mapped with the restriction enzymes Xma 1, Kpn 1, HindIII, Xba 1, Bgl I + II, and EcoRI. The locations of the sequences which code for 5 S, 5.8 S, 18 S, and 25 S ribosomal RNAs have been determined by hybridization of the purified RNA species with restriction endonuclease generated fragments of the repeating unit. The position of the 5.8 S ribosomal DNA sequences within the repeat was also established by sequencing the DNA which codes for 83 nucleotides at the 5' end of 5.8 S ribosomal RNA. The polarity of the 35 S ribosomal RNA precursor has been established by a combination of hybridization analysis and DNA sequence determination and is 5'-18 S, 5.8 S, 25 S-3'.

DNA↗
PubMed1977

Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.

Two new mutations are described which, together, eliminate essentially all the aminotransferase activity required for de novo biosynthesis of tyrosine, phenylalanine, and aspartic acid in a K-12 strain of Escherichia coli. One mutation, designated tyrB, lies at about 80 min on the E. coli map and inactivates the "tyrosine-repressible" tyrosine/phenylalanine aminotransferase. The second mutation, aspC, maps at about 20 min and inactivates a nonrespressible aspartate aminotransferase that also has activity on the aromatic amino acids. In ilvE- strains, which lack the branched-chain amino acid aminotransferase, the presence of either the tyrosine-repressible aminotransferase or the aspartate aminotransferase is sufficient for growth in the absence of exogenous tyrosine, phenylalanine, or aspartate; the tyrosine-repressible enzyme is also active in leucine biosynthesis. The ilvE gene product alone can reverse a phenylalanine requirement. Biochemical studies on extracts of strains carrying combinations of these aminotransferase mutations confirm the existence of two distinct enzymes with overlapping specificities for the alpha-keto acid analogues of tyrosine, phenylalanine, and aspartate. These enzymes can be distinguished by electrophoretic mobilities, by kinetic parameters using various substrates, and by a difference in tyrosine repressibility. In extracts of an ilvE- tyrB- aspC- triple mutant, no aminotransferase activity for the alpha-keto acids of tyrosine, phenylalanine, or aspartate could be detected.

Aspartate Aminotransferases↗
PubMed1977

Mapping of the aspartate and aromatic amino acid aminotransferase genes tyrB and aspC.

Co-transduction experiments using P1-mediated reciprocal and three-factor crosses have been used to map two mutations affecting the aspartate and aromatic amino acid aminotransferases of Escherichia coli. tyrB-, which inactivates the tyrosine-repressible component of these activities is co-transducible with metA and malB; the gene order is metA-malB-tyrB. aspC-, which inactivates the nonrepressible aminotransferase with high activity for aspartate, maps between and is co-transducible with serC and pyrD.

Aspartate Aminotransferases↗