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Biomedical subjects

M W Mueller

Publications and source records attributed to M W Mueller.

At least 19 recordsLinked to original sources
PubMed2003

Microarrayed recombinant allergens for diagnosis of allergy.

We suggest that the coapplication of recombinant allergens and microarray technology can lead to the development of new forms of multi-allergen tests which allow the determining and monitoring of complex sensitization profiles of allergic patients in single assays. The allergen extracts which have so far been used for diagnosis only allowed the determining of whether an allergic patient is sensitized against a particular allergen source, but the disease-eliciting allergens could not be identified. Through the application of recombinant DNA technology a rapidly growing panel of recombinant allergen molecules has become available which meanwhile comprises the epitope spectrum of most of the important allergen sources. We demonstrate that microarray technology can be used to establish multi-allergen tests consisting of microarrayed recombinant allergen molecules. Microarrayed recombinant allergens can be used to determine and monitor the profile of disease-eliciting allergens using single tests that require minute amounts of serum from allergic patients. The wealth of diagnostic information gained through microarray-based allergy testing will likely improve diagnosis, prevention and treatment of allergy.

Allergens↗
PubMed2001

Detection of single base alterations in genomic DNA by solid phase polymerase chain reaction on oligonucleotide microarrays.

DNA microarray technology holds significant promise for human DNA diagnostics. A number of technical approaches directed at the parallel identification of mutations or single nucleotide polymorphisms make use of polymerase-based specificity, like minisequencing or allele-specific primer elongation. These techniques, however, require separate laborious sample amplification, preparation, and purification steps, making large-scale analyses time and cost consuming. Here, we address this challenge by applying an experimental setup using simultaneous solid and liquid phase PCR on polyethyleneimine-coated glass slides, a novel microarray support allowing on-chip amplification reactions with exquisite specificity. A gene-specific oligonucleotide tiling array contains covalently attached allele-specific primers which interrogate single nucleotide positions within a genomic region of interest. During a thermal cycling reaction amplification products remain covalently bound to the solid support and can be visualized and analyzed by the incorporation of fluorescent dyes. Using the described procedure we unequivocally defined the presence of point mutations in the human tumor suppressor gene p53 directly from a natural DNA source. This semi-multiplex solid phase amplification format allowed the rapid and correct identification of 20 nucleotide positions from minute amounts of human genomic DNA. Our results suggest that this approach might constitute a vital component of future integrated DNA chip devices used in gene analysis.

Base Composition↗
PubMed2001

Mapping divalent metal ion binding sites in a group II intron by Mn(2+)- and Zn(2+)-induced site-specific RNA cleavage.

The function of group II introns depends on positively charged divalent metal ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+- and Zn2+-induced site-specific RNA cleavage to identify metal ions that fit into binding pockets within the structurally conserved bI1 group II intron domains (DI-DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in DI, two sites in DIII and two in DVI. All cleavage sites are located in the center or close to single-stranded and flexible RNA structures. Strand scissions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existence of Mg2+ binding pockets in physical proximity to the observed Mn2+-/Zn2+-induced cleavage positions. To distinguish between metal ions with a role in structure stabilization and those that play a more specific and critical role in the catalytic process of intron splicing, we combined structural and functional assays, comparing wild-type precursor and multiple splicing-deficient mutants. We identified six regions with binding pockets for Mg2+ ions presumably playing an important role in bI1 structure stabilization. Remarkably, assays with DI deletions and branch point mutants revealed the existence of one Mg2+ binding pocket near the branching A, which is involved in first-step catalysis. This pocket formation depends on precise interaction between the branching nucleotide and the 5' splice site, but does not require exon-binding site 1/intron binding site 1 interaction. This Mg2+ ion might support the correct placing of the branching A into the 'first-step active site'.

Binding Sites↗
PubMed2001

Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA.

Using reverse transcriptase-polymerase chain reaction (RT-PCR), we have recently described a bona fide deletion within the coding sequence of the large subunit of ribonucleotide reductase (R1) mRNA in colon cancer. Consecutive studies have raised questions about the nature of this phenomenon, because the corresponding genomic alteration at the DNA level or an aberrant protein could not be detected. Thus we considered an in vitro artifact during RT-PCR as a possible explanation for this observation. In contrast to reverse transcriptase, Taq DNA polymerase or C. therm DNA polymerase did not generate the aberrant product, suggesting the demand for the template switching activity intrinsic to retroviral reverse transcriptases. In fact, virtually the same deletion was observed in RT-PCR experiments when in vitro transcribed R1 mRNA was used. Considering structural prerequisites for template switching within R1 mRNA, we show that two direct repeats adjacent to a strong stem-loop secondary structure flank the deleted region of 1851 base pairs. Because several mRNAs encoding proteins of clinical and diagnostic importance fulfill these criteria, template switching enhances the potential risk of observing artifacts when interpreting results from RT-PCR studies. As shown in the present example, this may involve the artificial generation and the misinterpretation of PCR fragments amplified from targets relevant to tumor biology or cancer pharmacology. As a possible solution, one-step PCR with C. therm polymerase should be considered. This polymerase eliminates the artificial generation of aberrant mRNA signals observed during cDNA synthesis.

Adenocarcinoma↗
PubMed2000

Transposition and exon shuffling by group II intron RNA molecules in pieces.

In the realms of RNA, transposable elements created by self-inserting introns recombine novel combinations of exon sequences in the background of replicating molecules. Although intermolecular RNA recombination is a wide-spread phenomenon reported for a variety of RNA-containing viruses, direct evidence to support the theory that modern splicing systems, together with the exon-intron structure, have evolved from the ability of RNA to recombine, is lacking. Here, we used an in vitro deletion-complementation assay to demonstrate trans-activation of forward and reverse self-splicing of a fragmented derivative of the group II intron bI1 from yeast mitochondria. We provide direct evidence for the functional interchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result in trans-activation of intron transposition and RNA-based exon shuffling. The data extend theories on intron evolution and raise the intriguing possibility that naturally fragmented group III and spliceosomal introns themselves can create transposons, permitting rapid evolution of protein-coding sequences by splicing reactions.

Base Sequence↗
PubMed1999

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Here we present CapSelect as a novel experimental approach for the selective enrichment of full-length cDNAs in PCR-mediated analysis of mRNA sequences. The method combines the 5'-CAP-dependent addition of specifically three to four non-templated dCMP residues to the 3'-end of full-length cDNAs by reverse transcriptases in the presence of manganese and the controlled ribonucleotide tailing of cDNA ends by terminal deoxynucleotidyl transferase using rATP. By virtue of the generated terminal sequence motif (5'-dC(3-4)rA(3-4)), full-length cDNAs are selectively anchored to a double-stranded DNA adapter (with a dT(3-4)dG(3)3'-overhang) by T4 DNA ligase. The technique described is highly efficient, discriminates premature termination products and enriches full-length cDNAs.

Cell Line↗
PubMed1999

Customized perfusion circuit for the tiny ascending aorta.

Cannulating a pediatric patient with an interrupted aortic arch presents many challenges. Two cannulas are necessary to allow for proper blood flow to the head and to the extremities. This case was made more difficult by the weight of the child and the small size of the ascending aorta. The available pediatric cannulas were too large. The perfusionist adapted two 14-gauge intravenous (i.v.) catheters as arterial cannulas which were incorporated into the 1/4-inch arterial line. The 14-gauge i.v. catheters worked successfully. While on cardiopulmonary bypass, the patient was both metabolically and hemodynamically stable. The patient survived the procedure and was eventually discharged from hospital.

Aorta↗
PubMed1997

Transcription and activity of 5-fluorouracil converting enzymes in fluoropyrimidine resistance in colon cancer in vitro.

Cellular resistance to 5-fluorouracil (5-FU) is not completely understood. Since 5-FU shares the pyrimidine pathway with the physiological pyrimidines, we investigated the relationship between fluoropyrimidine metabolism, nucleic acid uptake and cytotoxicity of 5-FU in eight colon tumour cell lines including 5-FU-resistant subclones. The cytotoxicity of 5-FU was increased up to 423-fold when the anabolites 5-fluorouridine (FUrd), 5-fluorodeoxyuridine (FdUrd), and 5-fluorodeoxyuridine monophosphate (FdUMP) were compared with the parent drug in vitro. The enzymes uridine phosphorylase and thymidine phosphorylase were predictive for the cytotoxicity of 5-FU in 5/7 cell lines. Inhibition of uridine phosphorylase and thymidine phosphorylase by antisense strategies effectively antagonised 5-FU, abolishing 84% and 79% of its toxicity. The importance of thymidine phosphorylase was supported by a highly restricted enzyme activity in 5-FU-resistant cells. In 5-FU naive cells, a stimulating effect of 5-FU on thymidylate synthase mRNA and ribonucleotide reductase mRNA expression was observed. In these cells, antisense oligonucleotides to ribonucleotide reductase significantly reduced cell growth. Downregulation of ribonucleotide reductase mRNA in 5-FU-resistant subclones suggests different mechanisms in primary and secondary resistance to 5-FU. Most of the intracellular 5-FU was selectively incorporated into RNA (range: 45-91%) and generally spared DNA (range: 0.2-11%). In synthesising our data, we conclude that drug resistance could be overwhelmed through bypassing limiting steps in the activation of 5-FU. In the majority of colonic tumours, the activity of uridine phosphorylase and thymidine phosphorylase may have prognostic relevance for the cytotoxicity of 5-FU in vitro.

Antimetabolites, Antineoplastic↗
PubMed1997

DNA polymerization catalysed by a group II intron RNA in vitro.

The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.

Biopolymers↗
PubMed1997

Trans-activation of group II intron splicing by nuclear U5 snRNA.

Similarities between RNA splicing during autocatalytic excision of group II introns and pre-mRNA processing led to the hypothesis that group II introns might be the evolutionary predecessors of spliceosomal small nuclear RNAs. The ID3 subdomain stem-loop structure of group II introns, the proposed analogue of the spliceosomal U5 snRNA, is thought to be essential for 5' splice site recognition and anchoring of the free 5' exon. Using the group II intron bI1 we have analysed the role of ID3 in splicing. In the absence of ID3 the 5' splice site was recognized accurately and efficiently, but exon anchoring was greatly reduced. This step was restored in the presence of RNA fragments consisting of either the terminal stem-loop structure of ID3 or spliceosomal U5 snRNA. This suggests that the predominant role of both RNAs is to anchor the 5' exon during exon ligation. Furthermore, as U5 complements for the loss of ID3, a similar network of structural RNAs may form the catalytic core of both group II introns and spliceosomes.

Binding Sites↗
PubMed1996

Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase: a new approach in PCR-mediated analysis of mRNA sequences.

Controlled ribonucleotide tailing of cDNA ends (CRTC) by terminal deoxynucleotidyl transferase is a polymerase chain reaction (PCR)-mediated technique that was developed to facilitate cloning and direct sequence analysis of complete 5'-terminal unknown coding regions of rare RNA molecules. In contrast with standard tailing protocols using dNTPs as the substrate, ribo-tailing of cDNA ends is easily controllable, self-limited (from two to four rNMP incorporations) and highly efficient (>98%). By virtue of the homopolymeric ribo-tail, the modified cDNA is anchored to the 3' overhang of a double-stranded DNA-adaptor in a T4 DNA ligase-dependent ligation. PCR amplification, mediated by two sequence-specific primers, yields the desired unique product suitable for cloning and dideoxy-sequencing.

Base Sequence↗
PubMed1994

Transposable group II introns in fission and budding yeast. Site-specific genomic instabilities and formation of group II IVS plDNAs.

The recent report on RNA-mediated group II intron (IVS, intervening sequence) transposition in mitochondria (mt) of Saccharomyces cerevisiae and Podospora anserina and the demonstration of reverse transcriptase (RT) activity encoded by the mobile S. cerevisiae intron cox1-aI1 suggests that group II introns constitute a new class of site-specific retro-like (retroid) elements. This is supported by the finding that the mitochondrial cob1-bI1 intron from the fission yeast Schizosaccharomyces pombe, encoding an RT-like open reading frame, is transposed in mtDNA populations. In agreement with the involvement of an RNA-intermediate in IVS transposition: First, the insertion sites were preceded by at least an IBS1-like (intron binding site) motif, which corresponds to the upstream exon and suffices to form the IBS1/EBS1 (EBS: exon binding site) base-pairing interactions. Second, intron transposition was conservative with respect to sequences flanking the insertion sites. We formulated the hypothesis that transient IVS insertion at non-allelic sites followed by recombination can be viewed as a general molecular mechanism, applicable equally well to site-specific genomic instabilities involving splice-site borders of group II introns and to the formation of extra-genomic IVS plasmid DNAs (plDNAs). We used polymerase chain reaction (PCR) techniques to detect infrequent rearrangements in mtDNA and report here on duplicative IVS transposition, twintron formation (e.g. bI1 insertion into another bI1 intron), and IVS insertions at canonical 5' exon-intron borders in S. pombe (cob1-bI1) and in S. cerevisiae (cox1-aI1). These data substantiate the concept that group II intron homing, IVS transposition and circular IVS plDNA formation involve a common RNA-mediated mechanism. Finally, the findings suggest that extra-genomic group II IVS copies are not restricted to senescence mycelia of P. anserina, but constitute natural components of group II IVS-containing genomes.

Apoproteins↗
PubMed1993

Transposition of group II intron aI1 in yeast and invasion of mitochondrial genes at new locations.

Intron mobility at the RNA level by splicing reversal at allelic (homing) and non-allelic locations (transposition) has been reported in vitro. In the living cell, however, only intron homing by unidirectional gene conversion has been described. Supposing that intron insertions at non-allelic sites might occur in vivo, we speculated that group II splice-site-associated macro-deletions in fungal mitochondrial DNA might result from group II intron transposition to new locations followed by recombination. We used polymerase chain reaction techniques to detect this critical, infrequent intermediate in mtDNA populations. Here we report on group II intron aI1 transposition to non-allelic, splicing-compatible locations within the cox1 gene of yeast mtDNA. The identified integration sites are preceded by motifs similar to the upstream exon A1. Sequences flanking intron aI1 are not co-converted to the insertion sites and cis- and trans-acting mutations within aI1 reduce intron mobility below detection levels. These findings suggest the involvement of an RNA intermediate in group II intron transposition.

Alleles↗
PubMed1993

Group II intron RNA catalysis of progressive nucleotide insertion: a model for RNA editing.

The self-splicing bl1 intron lariat from mitochondria of Saccharomyces cerevisiae catalyzed the insertion of nucleotidyl monomers derived from the 3' end of a donor RNA into an acceptor RNA in a 3' to 5' direction in vitro. In this catalyzed reaction, the site specificity provided by intermolecular base pair interactions, the formation of chimeric intermediates, the polarity of the nucleotidyl insertion, and its reversibility all resemble such properties in previously proposed models of RNA editing in kinetoplastid mitochondria. These results suggest that RNA editing occurs by way of a concerted, two-step transesterification mechanism and that RNA splicing and RNA editing might be prebiotically related mechanisms; possibly, both evolved from a primordial demand for self-replication.

Adenosine↗
PubMed1985

Deletions in the cob gene of yeast mtDNA and their phenotypic effect.

Two cob- deletion mutants are characterized. One of them, M9410, is deleted for 911 bp of the noncoding sequences only which separate tRNAGlu and cob exon 1; it thus lacks most of the sequence encoding the 957 bp long cob leader (Bonitz et al. 1982) and some 20 bp 5' to it. The end points of this deletion coincide with 31 bp long direct repeats in wild type mtDNA. The other mutant, M9391, is deleted for all cob coding sequences and most of the cob leader sequence but it retains the 5' terminal 261 bp of this leader. Northern analysis revealed that M9410 totally lacks cob mRNA or pre-mRNA. The large deletion M9391 in contrast accumulates a 13S RNA which probably results from transcription through the junction, which ligates sequences of the cob leader to sequences of the cob-oli1 intergenic spacer.

Base Sequence↗