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D R Engelke

Publications and source records attributed to D R Engelke.

At least 55 records · Page 3Linked to original sources
PubMed1990

Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli.

DNA polymerase from Thermus aquaticus has become a common reagent in molecular biology because of its utility in DNA amplification and DNA sequencing protocols. A simplified method is described here for isolating the recombinant Taq enzyme after overproduction in Escherichia coli. Purification requires 8 to 10 h and entails heat treating and clearing the E. coli lysate, followed by precipitation of the enzyme with polyethyleneimine and elution from Bio Rex 70 ion exchange resin in a single salt step. The resulting enzyme preparation contains a single, nearly homogeneous protein consistent with the previously established size of the Taq DNA polymerase in a yield of 40-50 mg of protein per liter of cell culture.

Base Sequence↗
PubMed1990

ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences.

The ACE1 protein of Saccharomyces cerevisiae was expressed as a trpE-ACE1 fusion protein in Escherichia coli and shown to bind CUP1 upstream activation sequences at multiple regions in a copper-inducible manner. These binding sites contain within them the sequence 5'-TC(T)4-6GCTG-3', which we propose constitutes an important part of the ACE1 consensus recognition sequence.

Base Sequence↗
PubMed1989

Characterization of two active site mutations of thioredoxin reductase from Escherichia coli.

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.

Amino Acid Sequence↗
PubMed1989

Copper-induced binding of cellular factors to yeast metallothionein upstream activation sequences.

Copper-inducible transcription of the yeast metallothionein gene (CUP1) occurs by means of cis-acting upstream activation sequences (UAS) and trans-acting cellular factors. We have used a high-resolution chromosomal footprinting technique to detect the interaction of cellular factors with UASCUP1. Our results demonstrate that one or more cellular factors bind to UASCUP1 in a copper-inducible fashion. This copper-inducible binding is enhanced in a yeast strain that harbors several copies of the positive regulatory gene ACE1 and is not detectable in yeast cells that contain a nonfunctional (ace1-delta 1) locus. The correlation between yeast metallothionein gene activation and copper-inducible occupation of UASCUP1 sequences suggests that the binding of metallothionein transcriptional regulatory factors to cis-acting control sequences is copper-inducible.

Base Sequence↗
PubMed1989

Partial characterization of an RNA component that copurifies with Saccharomyces cerevisiae RNase P.

Saccharomyces cerevisiae cellular RNase P is composed of both protein and RNA components that are essential for activity. The isolated holoenzyme contains a highly structured RNA of 369 nucleotides that has extensive sequence similarities to the 286-nucleotide RNA associated with Schizosaccharomyces pombe RNase P but bears little resemblance to the analogous RNA sequences in procaryotes or S. cerevisiae mitochondria. Even so, the predicted secondary structure of S. cerevisiae RNA is strikingly similar to the bacterial phylogenetic consensus rather than to previously predicted structures of other eucaryotic RNase P RNAs.

Bacillus subtilis↗
PubMed1989

Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region.

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.

Base Sequence↗
PubMed1988

Initiation of rrn transcription in chloroplasts of Euglena gracilis bacillaris.

The site of initiation of chloroplast rRNA synthesis was determined by S1-mapping and by sequencing primary rRNA transcripts specifically labeled at their 5'-end. Transcription initiates at a single site 53 nucleotides upstream of the 5'-end of the mature 16S rRNA under all growth conditions examined. The initiation site is within a DNA sequence that is highly homologous to and probably derived from a tRNA gene-region located elsewhere in the chloroplast genome. A nearly identical sequence (102 of 103 nucleotides) is present near the replication origin. The near identity of the two sequences suggests a common mode for control of transcription of the rRNA genes and initiation of chloroplast DNA replication. The related sequence in the tRNA gene-region does not appear to serve as a transcript initiation site.

Animals↗
PubMed1988

Interaction of tRNA transcription factors with satellite I DNA from Xenopus laevis.

A cloned repeat of Xenopus laevis satellite I DNA was tested for the ability to form stable complexes with tRNA transcription factors in vitro. In template exclusion studies, the satellite I DNA competed efficiently with a tRNA gene for binding of yeast RNA polymerase III transcription factors. DNase I footprinting further showed that transcription factor TF IIIC alone bound to satellite I DNA at both the A block and B block consensus promoter sequences immediately downstream from the transcription start point. The strength and position of these associations indicate that satellite I DNA is a potential site for association of the same DNA-binding proteins that activate tRNA gene transcription.

Animals↗
PubMed1988

Direct sequencing of enzymatically amplified human genomic DNA.

The polymerase chain reaction is a recently described technique that uses flanking oligonucleotide primers and repeated cycles of enzymatic primer extension to amplify a short segment of DNA by greater than 100,000-fold. By use of sequencing primers located internal to the amplification primers, direct genomic sequence was obtained from enzymatically amplified DNA by using the dideoxynucleotide chain-termination method. The method is relatively simple and offers significant advantages in identifying mutations in genes for which the normal sequence is known. Heterozygous and homozygous mutations in the human beta- and gamma-globin loci were unambiguously identified in 3 days with less than 1 microgram of genomic DNA.

DNA↗
PubMed1987

Comparison of tRNA gene transcription complexes formed in vitro and in nuclei.

The nucleoprotein structure of single-copy tRNA genes in yeast nuclei was examined by DNase I footprinting and compared with that of complexes formed in vitro between the same genes and transcription factor C. Transcription factor C bound to both the 5' and 3' intragenic promoters of the tRNA(SUP53Leu) gene in vitro, protecting approximately 30 base pairs at the 3' promoter (B block) and 40 base pairs at the 5' promoter (A block) and causing enhanced DNase I cleavages between the protected regions. Binding to the two sites was independent of the relative orientation of the two sites on the helix and was eliminated by a single point mutation in the 3' promoter. The chromosomal tRNA(SUP53Leu) and tRNA(UCGSer) genes showed a pattern of protection and enhanced cleavages similar to that observed in vitro, indicating that the stable complexes formed in vitro accurately reflect at least some aspects of the nucleoprotein structure of the genes in chromatin.

Base Sequence↗
PubMed1986

Direct identification of small sequence changes in chromosomal DNA.

Dideoxynucleotide chain termination sequencing has been applied directly to genomic DNA templates by annealing radiolabeled oligodeoxynucleotide primers to unique sites in total yeast DNA and extending with avian myoblastosis virus (AMV) reverse transcriptase. The technique is used here to confirm the introduction of selectively altered tRNA genes into the Saccharomyces cerevisiae genome by gene replacement.

Base Sequence↗
PubMed1985

Nucleolytic processing of a tRNAArg-tRNAAsp dimeric precursor by a homologous component from Saccharomyces cerevisiae.

A subcellular extract from Saccharomyces cerevisiae has been used to transcribe cloned yeast tRNA genes in vitro and to process the primary transcripts at the 5' and 3' termini. Chromatographic fractionation of the extract has separated the transcription components from two distinct nucleolytic activities: an endonuclease that cleaves the precursors to produce mature 5' termini; and a 3'-5' exonuclease. These fractions have been used to elaborate a processing pathway for the dimeric primary transcript of the yeast tRNAArg-tRNAAsp gene pair. Under optimal conditions in vitro this gene is expressed at a rate of 200 transcripts/gene/hour, initiating at position -10 with respect to the mature 5' terminus of tRNAArg and terminating near position +160. The primary transcripts are cleaved by an endonuclease to give tRNAAsp with a mature 5' terminus, and a pre-tRNAArg monomer with a 5' leader and 3' trailer sequences. A second endonuclease cleavage of pre-tRNAArg generates the mature 5' terminus of tRNAArg. The endonuclease cleavages are not ordered. Exonuclease activity(ies) remove the spacer sequences from the 5' mature tRNAArg, and trim the 3' trailer portion from tRNAAsp. Exonucleolytic removal of the 3' trailer does not require prior endonuclease action, but removal of the spacer sequences from pre-tRNAArg is incomplete without prior removal of the 5' leader sequences.

Cell Nucleus↗
PubMed1984

HeLa cell RNA polymerase III transcription factors. Functional characterization of a fraction identified by its activity in a second template rescue assay.

That stable transcription complexes are formed in HeLa cell RNA polymerase III transcription extracts (Weil, P. A., Segall, J., Harris, B., Ng, S.-Y., and Roeder, R. G. (1979) J. Biol. Chem. 254, 6163-6173) can be shown in order of template addition experiments with DNAs coding for adenovirus 2 VA I RNA or one of the Bombyx mori tRNAAla2 genes. Using this property of the HeLa extracts in a "second template rescue assay" has allowed us to partially purify protein components involved in stable transcription complex formation. Two fractions, called transcription fraction X (TfrX) and transcription fraction Y (TfrY), are required to fully reconstitute selective transcription of the VA I and tRNAAla2 DNA templates. TfrX contains one or more components required for forming transcriptional pre-emptive complexes, as shown in order of addition experiments. TfrX strongly protects a DNA segment surrounding the highly conserved distal sequence (the so-called B block) of the VA I and tRNAAla2 genes from DNase I digestion; we have also characterized weak protection of other segments of these genes by TfrX. DNase I protection experiments with TfrX and probes prepared from deletion variants of the VA I gene show that an intact B block is required for the strong protection.

DNA-Directed RNA Polymerases↗
PubMed1984

Homologous in vitro transcription of linear DNA fragments containing the tRNAArg-tRNAAsp gene pair from Saccharomyces cerevisiae.

Transcription of a tRNAArg-tRNAAsp gene pair from Saccharomyces cerevisiae by an homologous yeast extract results in a dimeric percursor molecule which is processed to mature-sized tRNAArg and tRNAAsp molecules. We have transcribed linear DNA fragments cleaved within the gene sequences to show that precursor synthesis is not dependent on the internal promoter of the second gene (tRNAAsp). Furthermore, the second gene does not support independent transcription when the normal upstream initiation site is removed.

DNA, Fungal↗
PubMed1983

Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Rapid purification and structural and immunological properties.

DNA-dependent RNA polymerases I, II, and III (EC 2.7.7.6) were isolated from Xenopus laevis ovaries. The soluble enzymes were precipitated with polyethyleneimine and subjected to chromatography on heparin-Sepharose, DEAE-Sephadex, and phosphocellulose. RNA polymerase I was subjected to an additional chromatographic step on CM-Sephadex. The procedure required 40 h and produced purified RNA polymerase forms IA, IIA, and III in yields of 5 to 40%. The specific activities of RNA polymerases IIA and III (on native DNA) were comparable to those reported from other eukaryotic sources, whereas that of form IA was severalfold greater than the specific activities reported for other purified class I RNA polymerases. The complex subunit compositions of chromatographically purified RNA polymerases IA, IIA, and III were distinct when analyzed by polyacrylamide gradient gel electrophoresis under denaturing conditions, although all three classes contained polypeptides with Mr = 29,000, 23,000, and 19,000. Antibodies prepared against RNA polymerase III showed common antigenic determinants within the class I, II, and III enzymes. The sites responsible for the cross-reaction are located, at least in part, on the common 29,000-dalton polypeptide.

Animals↗