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T Christianson

Publications and source records attributed to T Christianson.

15 recordsLinked to original sources
PubMed1998

Etoposide sensitivity of radioresistant human glioma cell lines.

PURPOSE: Malignant gliomas display aggressive local behavior and are not cured by existing therapy. Etoposide, a topoisomerase-II-inhibitor agent, is one of the most active and useful antineoplastic agents. However, etoposide is not usually used on these tumors. We undertook an in vitro study to prove that etoposide is a useful drug for malignant gliomas. METHODS: Five human glioma cell lines were the basis for this study. Following exposure to various concentrations of etoposide, the glioma cell lines were found to be sensitive; the median concentration inhibiting the number of cells by 50% (IC50) was 8.76 microg/ml (range 8-15.8 microg/ml). Since topoisomerase II is the critical target for etoposide, it was of interest to determine the topoisomerase II activity (decatenation of kinetoplast DNA isolated from Cryphtidia fasciculata) and the etoposide-induced inhibition of topoisomerase II activity. RESULTS: The topoisomerase II activity was homogeneous in glioma cell lines (average of 50% decatenation with 7,000 cells), and topoisomerase II was the target of the etoposide. CONCLUSIONS: Our results suggest that topoiomerase II-reactive agents may prove to be clinically useful drugs for patients with malignant gliomas.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Unusual ligand binding at the active site domain of an engineered mutant of subtilisin BL.

As an attempt to recruit the third calcium binding site of thermitase into subtilisin BL, a Bacillus lentus alkaline protease (BLAP), the amino acid sequence from position 50 to 60 and position 92 was modified to the equivalent amino acids in thermitase. The resulting protein, designated BLAPm109, exhibited unusual biochemical features. Peptide mapping and gel electrophoresis revealed that two protein species co-purify in a ratio of about 1:1. Form 1 consisted of a single polypeptide of 269 amino acid residues. Form 2 was the same protein but with an internal peptide bond cleavage at the C-terminus of position 54. On electropherograms a dimer of Form 1 and Form 2 was also detectable. A zymogram showed that all three molecular species were catalytically active. From this protein mixture, crystals suitable for X-ray analysis were nevertheless obtained. SDS-PAGE of protein recovered from a crystal revealed that only Form 2 appears. in the crystal. The space group for this crystal was P21 with unit cell dimensions of a=42 angstroms, b=58 angstroms, c=47 angstroms and beta = 106.3 degrees. Examination of the preliminary electron density map revealed that the "thermitase loop" from 50 to 60 departs from the surface of the protein and winds through the active site of a symmetry-related copy of the asymmetric unit.

Amino Acid Sequence↗
PubMed1995

SKOV3 ovarian carcinoma cells have functional estrogen receptor but are growth-resistant to estrogen and antiestrogens.

Estrogen receptor positive ovarian cancer is often refractile to antiestrogen therapy. Here we describe the SKOV3 human ovarian carcinoma cell line as an in vitro model for estrogen and antiestrogen resistant ovarian cancer. While SKOV3 cells expressed estrogen receptor (ER) mRNA and protein at a similar level as the estrogen responsive T47D breast carcinoma cell line, their growth was not responsive to estradiol (E2) and was not inhibited by the antiestrogens OH-tamoxifen and ICI 164,384. The ER in SKOV3 cells was normal with respect to apparent Kd for binding with E2, E2 regulation of a transiently transfected ERE driven reporter gene, and E2 stimulation of expression of the early growth response genes c-myc and c-fos. However, the SKOV3 cells exhibited no expression of the progesterone receptor gene (PR) even after addition of E2, and the protein products of the estrogen responsive genes HER-2/neu and cathepsin D were expressed at constitutive levels that were not regulated by E2. Therefore, estrogen resistance in these cells may be a result of constitutive expression and loss of E2 regulation of selected growth regulatory gene products rather than a defect in estrogen activation of ER as a transcriptional regulator.

Blotting, Northern↗
PubMed1994

Peptide mapping of subtilisins as a practical tool for locating protein sequence errors during extensive protein engineering projects.

Protein engineering based on rational design is an iterative process of sequential amino acid residue replacements. This requires a rapid and sensitive method for checking the protein sequence after each round of mutagenesis. As shown with subtilisin BL, acid treatment followed by urea denaturation renders the enzyme degradable by trypsin within 10 min. Separation of the peptides by reversed-phase HPLC produces a map that differentiates even the most conservative alteration on peptides as large as 48 amino acid residues. The method was used to uncover erroneous mutations; to determine the concentration of active protease relative to an internal standard of known specific activity; to measure the rate of oxidation of methionine-216 in the oxyanion hole of subtilisin BL; and to document that under these conditions no other methionine in the molecule is oxidized by hydrogen peroxide.

Amino Acid Sequence↗
PubMed1993

Zymogram of proteases made with developed film from nondenaturing polyacrylamide gels after electrophoresis.

A simple, extremely versatile method for preparing zymograms from proteases after nondenaturing Phast-System gel electrophoresis is described. After completion of the run and before staining, an electropherogram and a piece of developed, single-side coated X-ray film are brought into contact for 5 min at room temperature. The film overlay is then separated and the gel is stained for protein. The zymogram on the X-ray film is generated by simply pouring about 10 ml of a suitable buffer solution at 30 to 50 degrees C over the film strip. Clearing zones appeared within a few seconds to a few minutes depending on protease amount. For the alkaline protease subtilisin BL the lower limit of detectability was 10 ng applied to the gel prior to electrophoresis. For preservation and archiving the zymogram film is simply rinsed with distilled water and air-dried. The film can be cut to size and mounted for a slide projector, or the film can serve as a negative for photographic enlargements. The clearing zone area is proportional to the amount of protease from 10 to 100 ng of protein.

Bacillus subtilis↗
PubMed1993

Strategy and implementation of a system for protein engineering.

This paper describes an overall view of an industrial protein engineering project from conception to successful completion. The choice of rational design was determined by the availability of an excellent three-dimensional crystal structure and the availability of information in the literature to define a strategy. The design strategy was refined extensively during the course of the project. The development of methods for mutagenesis, expression, verification, purification, and characterization of mutant enzymes is dictated in part by the enzyme property one chooses to modify and must be rapid yet accurate. Such an approach would be applicable to improve the stability of any other protein or enzyme. Using this approach, we successfully increased the stability of subtilisin BL over 10-fold at 50 degrees C with an overall success rate greater than 60%.

Bacillus↗
PubMed1983

Initiation of transcription of the yeast mitochondrial gene coding for ATPase subunit 9.

We have determined transcriptional initiation sites for the ATPase subunit 9 gene on the yeast mitochondrial genome. Using S1 nuclease mapping, in vitro capping of primary transcripts with GTP and guanylyl transferase, and in vitro transcription analysis with purified mitochondrial RNA polymerase, we find the major site of transcriptional initiation to be at a point 630 nucleotides upstream of the coding region for the gene. In addition, we find much lower levels of initiation at a second site 78 nucleotides downstream of the first. Both initiation sites occur at the same position within a nonanucleotide sequence which we have previously found associated with initiation of rRNA synthesis. This work further supports the notion that this nonanucleotide sequence is an integral component of mitochondrial promoters and indicates that the same RNA polymerase is used for transcription of both mRNA and rRNA in yeast mitochondria.

Adenosine Triphosphatases↗
PubMed1983

Identification of multiple transcriptional initiation sites on the yeast mitochondrial genome by in vitro capping with guanylyltransferase.

We have studied transcriptional initiation in the mitochondria of the yeast Saccharomyces cerevisiae by analyzing mitochondrial transcripts from grande and petite yeast after labeling in vitro with vaccinia virus guanylyltransferase and [alpha-32P]GTP. This procedure labels triphosphate-terminated RNA which arises from transcriptional initiation. Exploiting the extremely low GC content (18%) of yeast mitochondrial DNA, we digested the in vitro capped transcripts with the G-specific ribonuclease T1; this resulted in 27 oligonucleotides varying in size from 2 to 51 nucleotides. RNA from 14 overlapping petites was analyzed and 20 transcripts were localized by deletion mapping. Nineteen oligonucleotides were sequences and 13 were identified and precisely localized by comparison with known DNA sequences. In all cases, transcription is initiated at a consensus nonanucleotide sequence which can be considered part of the yeast mitochondrial promoter. We identified initiation sites for the 21 S and 14 S rRNAs; the phenylalanine, f-methionine, and glutamic tRNAs; two sites for the OLI-1 gene; and three for the ori (rep) regions. Most promoters appear to give rise to very long multigene primary transcripts. Examples are multigene transcripts for the glutamic tRNA and COB genes and for the OLI-1, serine tRNA, and Var genes. Since the consensus nonanucleotide sequences at the ori regions are similar to those at other transcriptional initiation sites, it is likely that the same RNA polymerase primes DNA replication and gene transcription.

Base Sequence↗
PubMed1983

Identification of a single transcriptional initiation site for the glutamic tRNA and COB genes in yeast mitochondria.

We have identified a single transcriptional initiation site for the glutamic tRNA and COB (cytochrome b) genes by using the complementary techniques of in vitro capping of RNA and in vitro transcription. In the capping reaction, mitochondrial RNA is labeled with [alpha-32P]GTP by vaccinia virus guanylyltransferase. This reaction is specific for the 5' ends of RNA retaining the terminal triphosphate of transcriptional initiation. Exploiting the extremely low G+C content (18%) of yeast mitochondrial DNA, we digested in vitro capped transcripts from various petite deletion mutants with the G-specific RNase T1. By petite deletion mapping, a capped transcript giving rise to a 51-base RNase T1-generated oligonucleotide was localized near the glutamic tRNA gene. When the sequence of this oligonucleotide was determined, it perfectly matched the DNA sequence 391 base upstream of the glutamic tRNA. Purified yeast mitochondrial RNA polymerase initiated transcription in vitro at the same site as shown by the sequence of the 33-base oligonucleotide product of the reaction performed in the absence of CTP. Initiation starts at a nonanucleotide sequence previously implicated in yeast mitochondrial transcriptional initiation. Because there is no evidence of an initiation site in the 1,050 bases between the glutamic tRNA and COB genes, the two genes are likely to be transcribed together. Further evidence of a long common transcript was provided by RNA blot hybridization.

Base Sequence↗
PubMed1982

A nonanucleotide sequence involved in promotion of ribosomal RNA synthesis and RNA priming of DNA replication in yeast mitochondria.

We have examined the initiation of transcription of the mitochondrial genes for ribosomal RNA (rRNA) in the yeast Kluyveromyces lactis and show that these are transcribed independently from individual promoters. The mature large rRNA contains a 5' di- or triphosphate end which can be labelled in vitro with [alpha-32P]GTP using guanylyltransferase and this enabled us to determine the nucleotide sequence of its 5' terminus. For the small rRNA, a minor in vitro capped RNA species hybridizes in the region where--as judged from S1 nuclease protection experiments--the precursor of this RNA starts. We have determined the DNA sequence around the beginning of both rRNA genes and this reveals the existence of an identical nonanucleotide sequence (5' -ATATAAGTA- 3') just preceding the positions where the rRNAs start. This sequence is identical to the one preceding the rRNA genes in the mtDNA of the distantly related yeast Saccharomyces cerevisiae (Osinga, K.A. and Tabak, H.F. (1982) Nucl.Acids Res. 10, 3617-3626) and supports our proposal that this sequence motif is part of a yeast mitochondrial promoter. We have noticed that the same sequence is located in the putative origin of replication present in hypersuppressive petite mutants of S. cerevisiae and consider the possibility that this sequence is involved in RNA priming of DNA replication.

Ascomycota↗
PubMed1982

Transcriptional initiation and processing of the small ribosomal RNA of yeast mitochondria.

We have identified the nucleotide at which transcription initiates on the yeast mitochondrial small (14 S) rRNA gene by sequencing of RNA labeled at the 5' initiating triphosphate with vaccinia virus guanylyltransferase [alpha-32P]GTP (in vitro capping reaction). Initiation occurs within the stem of a 12-base palindromic repeat. The initiation sequence has homology with the large (21 S) ribosomal RNA initiation sequence that has been previously determined. We have also sequenced the 5' and 3' ends of the mature 14 S rRNA after labeling with T4 polynucleotide kinase and RNA ligase, respectively. These sequences demonstrate that about 80 nucleotides are cleaved from the 5' end of a precursor to produce the mature 14 S rRNA. This cleavage is imprecise in that the processing occurs at one of five adjacent nucleotides 77 to 81 nucleotides downstream from the 5' initiation site. The 3' ends of this precursor and the mature 14 S rRNA are unique and identical.

Base Sequence↗
PubMed1982

Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA.

The initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mitochondrial DNA have been investigated by mapping experiments utilizing in vitro "capped" mitochondrial RNA molecules or nascent RNA chains. Mitochondrial poly(A)-containing RNA molecules were labeled at their 5' ends with [alpha-32P]GTP and guanylyltransferase ("capping" enzyme) and mapped on the mitochondrial genome by DNA transfer hybridization and S1 nuclease protection experiments. A mapping site for the capped 5' ends was found on the H strand very near to the 5' terminus of the 12S rRNA gene, and another site was found on the L strand very near to the 5' terminus of the 7S RNA coding sequence. In parallel experiments, the 5' ends of the nascent chains isolated from mitochondrial DNA transcription complexes were similarly mapped very near to the 5' termini of the 12S rRNA gene and of the 7S RNA coding sequence. The in vitro capped RNA molecules and the nascent chains thus presumably identify the same transcriptional initiation sites on the H strand and the L strand. The occurrence of a second possible initiation site for H-strand transcription 90-110 nucleotides upstream of that described above--i.e., 20-40 nucleotides upstream of the tRNAPhe gene--had been previously indicated by a mapping analysis of the nascent RNA chains and has been confirmed in the present work. The presence of two initiation sites for H-strand transcription can be correlated with other types of evidence that point to two different transcription events leading to the synthesis of a polycistronic molecule corresponding to the almost entire H strand and to the synthesis of the rRNA species.

Chromosome Mapping↗
PubMed1980

Processing of precursors of 21S ribosomal RNA from yeast mitochondria.

The transcription and processing of mitochondrial 21S rRNA in a petite strain of Saccharomyces cerevisiae has been examined by electron microscopic analysis of R-loop hybrids and by hybridization of labeled mitochondrial DNA probes to RNA transferred to diazobenzyloxymethyl paper. We have shown the presence of a large [5.1- to 5.4-kilobase (kb)] transcript that appears to be a precursor of mitochondrial 21S rRNA. This transcript contains sequences homologous to those of the mature 21S rRNA, to the intervening sequence present in the gene, and to additional sequences at the 3' end of the molecule. Our data suggest that this precursor of 21S rRNA is processed in two steps. The intron sequence is usually excised first, followed by removal of the extra 3' sequences. In some cases, however, the 3' extension is first removed and the intron sequence is then excised. Both pathways appear to lead to formation of the 3.1-kb mature 21S rRNA and a stable 1.2-kb intron transcript. Similar results were obtained with grande MH41-7B mitochondrial RNA by RNA transfer hybridization. We have also observed a number of additional transcripts that may be normal processing intermediates or may result from faulty cleavage-ligation during excision of the intervening sequence.

Chromosome Mapping↗
PubMed1979

Propagation of restriction fragments from the mitochondrial DNA of Saccharomyces cerevisiae in E. coli by means of plasmid vectors.

Some of the EcoRI fragments of yeast (Saccharomyces cerevisiae) mitochondrial DNA were cloned into E. coli using plasmid pMB9. The five smallest fragments in molecular weight appeared to be preferentially retained by E coli; partial fragments derived from larger mitochondrial DNA fragments were also found. One of the fragments, R7 (2.4 kb), may contain the OII gene. Cloned R7 DNA was stable under a variety of growth conditions, but showed some changes in molecular weight after transfer to different E. coli strains. Fragment R7 is transcribed in minicells, producing RNA that hybridizes specifically to mitochondrial DNA. Both DNA strands are transcribed, in contrast to the asymmetric transcription found in mitochondria. No new polypeptides were observed in minicells containing cloned fragment 7.

DNA Restriction Enzymes↗