Biomedical subjects

C Schmelzer

Publications and source records attributed to C Schmelzer.

At least 19 recordsLinked to original sources

PubMed1992

New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates.

Here we describe three novel reactions of the self-splicing group II intron bI1 (the first intron of the COB gene of yeast mitochondria) demonstrating its catalytic versatility: reversal of the first step of the self-splicing reaction catalyzed by a linear form of the intron utilizing the energy of a phosphoanhydride bond for transesterification, ligation of a single-stranded DNA to an RNA, and cleavage of a single-stranded DNA substrate. These results have the following evolutionary implications: use of the alpha-beta bond of a terminal triphosphate for transesterification suggests that an RNA RNA replicase could use mononucleotide triphosphates as precursors, and cleavage of single-stranded DNA and DNA-RNA ligation suggests that excised group II introns might integrate directly into DNA without prior reverse transcription.

Amino Acid Sequence↗

PubMed1991

Restoration of the self-splicing activity of a defective group II intron by a small trans-acting RNA.

The yeast mitochondrial group II intron bI1 is self-splicing in vitro. We have introduced a deletion of hairpin C1 within the structural domain 1 that abolishes catalytic activity of the intron in the normal splicing reaction in cis, but does less severely affect a reaction in trans, the reopening of ligated exons. Since exon reopening is supposed to correspond to a reverse 3' cleavage this suggests that the deletion specifically blocks the first reaction step. The intron regains its activity to self-splice in cis by intermolecular complementation with a small RNA harbouring sequences lacking in the mutant intron. These results demonstrate the feasibility to reconstitute a functionally active structure of the truncated intron by intermolecular complementation in vitro. Furthermore, the data support the hypothesis that group II introns are predecessors of nuclear pre-mRNA introns and that the small nuclear RNAs of the spliceosome arose by segregation from the original intron.

Base Sequence↗

PubMed1991

Structural requirements for selection of 5'- and 3' splice sites of group II introns.

The group II intron bl1 in the gene for apocytochrome b in yeast mitochondrial DNA (COB) is self-splicing in vitro. It could recently be shown that self-splicing of this intron is fully reversible in vitro. In addition, intron integration is not restricted to parental exons, since the intron can also integrate into a foreign RNA. The position of insertion seems to be immediately 3' to a cryptic intron binding site 1 (IBS1). We confirmed and extended these results by sequencing 26 individual RNAs with transposed introns after reverse transcription and PCR amplification. Results show that intron integration into authentic exons is generally correct, but that integration into a foreign RNA is often inaccurate, i.e. insertion is one nt downstream or upstream of the 3' end of IBS1. This leads to the generation of 5' splice junctions of the new intron-harbouring 'preRNAs' with addition (or deletion) of a single A residue at the 3' end of IBS1. To investigate which structures help to define the position of 5'- and 3' cleavage, preRNAs of i) these clones with aberrant 5' splice junctions and ii) preRNAs with artificial hairpins between domains 5 and 6 of the intron were spliced under different reaction conditions. Results obtained let us conclude that i) branchpoint dependent 5' cleavage is directed by the 5' terminal G residue of the intron and, ii) the first nucleotide(s) of the 3' exon play an important role in defining the 3' splice site.

Apoproteins↗

PubMed1991

[Development of a spacer for inhaled corticosteroid. Evaluation using flunisolide metered dose aerosol].

As optimal therapy with inhaled steroids invariably demands the use of a spacer (Nolte, 1989), a specific inhalation device adapted to flunisolide metered dose inhaler was required. For this purpose, various inhalation chambers varying in geometry and material were investigated. The decisive criterion was the percentage of inhalable particles (diameter less than 8 microns) dispensed by the device. Separation of larger particles was possible in spacers with a relatively small volume. The highest dispensing rate being achieved with conical or pear-shaped inhalation chambers. An increase in spacer volume beyond 320 ml failed to improve the percentage of inhalable active substance. The results demonstrate that the spacer, dimensioned to the smallest possible volume, specially developed for Inhacort MDI has optimal dispensing characteristics for flunisolide.

Adrenal Cortex Hormones↗

PubMed1991

Generation of polyclonal antibodies against recombinant human activin A.

A goat antiserum to purified recombinant human activin A (rhAct-A), a dimer formed by two beta A-subunits of inhibin, has been produced. The immunoreactivity of the antiserum has been evaluated in an antigen coated enzyme-linked immunosorbent assay, in a radioimmunoassay using iodinated rhAct-A, and by Western blot analysis. The antiserum demonstrated some cross reactivity to inhibin A, a structurally related heterodimer which contains an identical beta A-subunit coupled to a distinct, though similar, alpha subunit. A simple radioimmunoassay for rhAct-A in tissue culture supernatant has been developed with rhAct-A affinity column purified polyclonal antiserum. The assay is precise and sensitive with a range of 0.31-40 ng/ml. The cross reactivity of inhibin A in the RIA is about 4.3%. Despite its cross-reactivity this antiserum will facilitate studies of the physiology of activin A and inhibin A which includes a Western blot analysis where a molecular size distinction is accomplished.

Activins↗

PubMed1990

Group II intron RNA-catalyzed recombination of RNA in vitro.

We report the first evidence for a novel reaction mediated by the self-splicing yeast mitochondrial group II intron bl1; the site-specific recombination of RNA molecules in vitro. Upon incubation of the intron lariat with two different RNAs, each harbouring a short sequence complementary to exon binding site 1 (EBS1) of the intron, novel recombined RNAs are formed. As a result of this intron-mediated shuffling of gene segments, the 5' part of RNA1 is ligated to the 3' part of RNA2 and, reciprocally, the 5' part of RNA2 to the 3' part of RNA1. Sequence analysis of the recombinant junction shows that the site of recombination is precisely located 3' to intron binding site 1 (IBS1). The hypothesized mechanism of recombination involves exchange of RNA 5' parts after the first step of a reverse splicing reaction. The possible role of this mechanism in vivo and during prebiotic evolution is discussed.

Base Sequence↗

PubMed1990

Effect of deletions at structural domains of group II intron bI1 on self-splicing in vitro.

Some group II introns can undergo a protein-independent splicing reaction with the basic reaction pathway similar to nuclear pre-mRNA splicing and the catalytic functions of some of the structural components have been determined. To identify further functional domains, we have generated an ensemble of partial and complete deletions of domains I, II, III and IV of the self-splicing group II intron bI1 from yeast mitochondria and studied their effects on the splicing reaction in vitro. Our results indicate that domains II and IV, which vary considerably in length and structure among group II introns, do not play a direct role in catalysis but mainly help to ensure the proper interaction between upstream and downstream catalytically active structural elements. Deletions of sub-domains of domain I and domain III indicate that these elements are involved in 5' cleavage by hydrolysis and in a reaction in trans (exon reopening), and that this function can be inhibited without affecting the normal 5' cleavage by transesterification. Yet, we infer that the helical structures affected by the mutational alterations might not contribute to this reaction mode per se but that changes within local secondary structures perturb the internal conformation of the ribozyme. Furthermore, we have designed an abbreviated version of intron bI1, with a length of 542 nucleotides, which is still catalytically active.

Base Sequence↗

PubMed1990

Integration of group II intron bI1 into a foreign RNA by reversal of the self-splicing reaction in vitro.

Group II intron bI1, the first intron of the COB gene in the mitochondria of S. cerevisiae, is able to self-splice in vitro with the basic pathway similar to nuclear pre-mRNA splicing. We show that incubation of the intron lariat with ligated exons bE1 and bE2 leads to a complete reversal of the splicing reaction. The integration of the intron into the ligated exons is correct; the reconstituted preRNA of the reverse reaction can undergo a self-splicing reaction anew. When incubated with a foreign RNA species bearing a sequence motif that is complementary to exon binding site 1, the lariat can integrate into this RNA with the position of insertion immediately downstream of this sequence. This result implies that transposition of group II introns on the RNA level by reversal of the splicing reaction is, in principle, conceivable.

Base Sequence↗

PubMed1990

Self-splicing of the mobile group II intron of the filamentous fungus Podospora anserina (COI I1) in vitro.

The first intron of the mitochondrial gene coding for cytochrome oxidase subunit I (COI I1) of Podospora anserina can undergo self-splicing in vitro at high concentrations of NH4Cl or KCl. Under these conditions cleavage at the 5' splice junction takes place without branch formation probably via hydrolysis by water or OH- and the intron is released in a linear form. In vitro transcripts that contain mutated introns with large deletions in nonconserved domain IV comprising greater than 50% of the intronic sequence display a more efficient splicing reaction and, surprisingly, 5' cleavage via transesterification and lariat formation is re-established to a low degree under NH4Cl. In contrast to the self-splicing group II introns aI5 gamma and bI1 from yeast mitochondria cleavage at the 3' splice site of the Podospora intron is reduced and cleavage by hydrolysis in trans (i.e. exon reopening) is almost completely suppressed. Both observations could be interpreted as a result of unfavourable spatial conformations of the intron that (i) lead to a steric hindrance of the 5' exon to attack the 3' splice site in cis and (ii) block intron-dependent cleavage reaction of the ligated exons in trans. Alternatively, the possibility that a weak overall interaction of the postulated exon- with the corresponding intron-binding sites (EBS-IBS pairings) is responsible for the remarkable differences to the self-splicing reaction of other group II introns is discussed.

Ascomycota↗

PubMed1988

Selection of cryptic 5' splice sites by group II intron RNAs in vitro.

Recognition of 5' splice points by group I and group II self-splicing introns involves the interaction of exon sequences--directly preceding the 5' splice site--with intronic sequence elements. We show here that the exon binding sequences (EBS) of group II intron aI5c can accept various substitutes of the authentic intron binding sites (IBS) provided in cis or in trans. The efficiency of cleavages at these cryptic 5' splice sites was enhanced by deletion of the authentic IBS2 element. All cryptic 5' cleavage sites studied here were preceded by an IBS1 like sequence; indicating that the IBS1/EBS1 pairing alone is sufficient for proper 5' splice site selection by the intronic EBS element. The results are discussed in terms of minimal requirements for 5' cleavages and position effects of IBS sites relative to the intron.

Exons↗

PubMed1987

Self-splicing of group II introns in vitro: lariat formation and 3' splice site selection in mutant RNAs.

Deletion or substitution of the branch A residue in group II intron bl1 significantly reduces splicing activity; yet, residual exon ligation is correct, and lariats have their branch points at the normal distance from the 3' end of the intron. Mutations in the sequence facing the branch point also allow residual lariat formation; however, free 3' exons are generated with false 5' termini, all of which are within a UCACA consensus sequence located upstream or downstream of the normal 3' splice site. These results indicate that both the conserved 3' splice site APy and the spatial arrangements in stem 6 are crucial for correct 3' splice site selection.

Electrophoresis, Polyacrylamide Gel↗

PubMed1987

Amplification of the yeast nuclear gene MRS3 confers suppression of a mitochondrial RNA splice defect.

The MRS3 gene cloned in the multicopy plasmid YEp13 suppresses the mitochondrial splice defect exerted by mutation M1301 in the group II intron bI1. In this article we report on the behavior of the MRS3 gene cloned in the integration vector pEMBLYi27 and in the CEN4-ARS vector YCp50. Transformation of mutant M1301 cells with these recombinant vectors produced transformants, the majority of which showed the original splice defect and contained the recombinant vectors in single or low copy; a minority, however, was splicing competent and showed exceptionally high copy numbers of the MRS3 gene. These latter transformants had either the pEMBLYi27/MRS3 sequence repeated at least 20 times in tandem at the chromosomal site of the MRS3 gene or they had the YCp50/MRS3 sequence established as a multicopy plasmid lacking the copy number control usually exerted by the CEN4 sequence in this plasmid.

Cloning, Molecular↗

PubMed1987

Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number.

A gene bank of a yeast wild type DNA in the high copy number vector YEp13 was screened for recombinant plasmids which suppress the mitochondrial RNA splice defect exerted by mutant M1301, a -1 bp deletion in the first intron of the mitochondrial COB gene (bI1). A total of 17 recombinant plasmids with similar suppressor activity were found. Restriction mapping and cross-hybridization of the inserts revealed that these 17 plasmids contain three different inserts, all lacking any extended sequence homology. Each of the inserts, when present in high copy number, has a similar suppressor activity: high in the presence of mutation M1301 in bI1, a group II intron, and low but significant with the presence of few mutants in bI2 and bI3 of the COB gene, both of which are group I introns.

Alleles↗

PubMed1986

Self-splicing of group II introns in vitro: mapping of the branch point and mutational inhibition of lariat formation.

Group II intron bl1 from yeast mitochondria can undergo self-splicing in vitro. Exons become correctly ligated, and the excised intron has a lariat structure similar to that of introns from nuclear mRNA. The branch point of the bl1 lariat is located eight or nine nucleotides upstream of the 3' end of the intron and is part of a hairpin structure that is well conserved among group II introns. Several mutations next to the branch point and in other parts of the core structure of group II introns are shown to affect lariat formation. One of them, carried by strain M4873, abolishes splicing in vivo and in vitro, apparently by changing the architecture of the hairpin structure containing the branch point. Similarities between group II introns and nuclear pre-mRNA introns are discussed in terms of evolutionary relatedness.

Base Sequence↗

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↗

PubMed1983

Determination of functional domains in intron bI1 of yeast mitochondrial RNA by studies of mitochondrial mutations and a nuclear suppressor.

The sequence of intron 1 in the cob gene in mtDNA (bI1) of the yeast strain 777-3A has been determined. Furthermore, we have performed a systematic search for complementary sequence stretches within this intron RNA, and within the RNA of intron 5 gamma of the oxi3 gene (aI5 gamma) which shares distinctive sequences with bI1. Possible secondary structure models derived from this analysis show nearly identical core structures for bI1 and aI5 gamma RNA with conserved sequence stretches in prominent positions. These core structures are similar to those previously reported for RNAs of introns having very limited sequence homology with bI1 and aI5 gamma. In two mutants which are defective in bI1 excision from cob pre-mRNA, nucleotide sequence alterations in bI1 have been determined. One mutation (G5049) apparently affects the stability of a hybrid stretch in the proposed secondary structure of bI1 RNA whereas the other one (M1301), a deletion of one A in a run of five As, affects a sequence which is conserved in bI1 and aI5 gamma and is involved in the formation of a distinct secondary structure. Out of seven revertants of M1301, three were found to have restored the wild-type bI1 sequence AAAAA, three others had the related sequence AAAAG which is functionally indistinguishable from wild-type, whereas one revertant had a nuclear mutation which suppresses the splicing defect exerted by the mitochondrial mutation M1301. This nuclear suppressor (SUP-101) is allele specific and dominant. The possible role of the sequence affected by M1301 in terms of a recognition site for a nuclear gene product will be discussed.

Base Sequence↗

PubMed1982

Identification of splicing signals in introns of yeast mitochondrial split genes: mutational alterations in intron bI1 and secondary structures in related introns.

Four mitochondrial mutations are known to block excision of intron I1 of the cob gene in S.cerevisiae. The nucleotide sequence alteration of one of them, M4873, has been determined. It is a deletion of 1 bp in a run of five G's at a distance of 30 to 34 bp upstream to the 3' splice point. Reversion is found to occur by restoration of the run of five G's either by insertion of 1 G (wild type reversion) or by transition A leads to G next to this run of G's (pseudo-wild type reversion). The effect of mutation and reversion on RNA splicing indicates that the run of five G's is of critical importance for intron I1 excision, possibly in participating in the formation of a splice signal with a helical structure. This presumption is confirmed by the observation that this sequence is part of a larger sequence of some 80 bp next to the 3' splice point which is conserved to some extend in the four mitochondrial introns (bI1, aI1, aI2, aI5) that survive after excision as circular RNAs. Most striking is the conservation of this sequence at the level of secondary structure.

Base Sequence↗

PubMed1982

Evidence for ribosomes involved in splicing of yeast mitochondrial transcripts.

We have investigated the processing of transcripts of the split gene COB in yeast mitochondrial DNA from cells whose mitochondrial translation was blocked by chloramphenicol for several generations of cell growth. First analysis of transcripts by electrophoresis and RNA/DNA-hybridization clearly showed that cell growth in the presence of CAP leads to an inhibition of processing yielding an increasing amount of splicing intermediates of the COB transcript and decreasing amounts of the 18S mRNA coding for apocytochrome b. This observation is in accordance with the now widely favoured idea that mitochondrial proteins are involved in splicing of COB transcripts and that their reduction should hamper processing and - therefore - lead to an accumulation of pre-mRNAs. However, further information obtained by pulse-labeling of pre-mRNA in vivo in the presence of CAP for various times shows that even 30 minutes after addition of CAP a reduction of the processing rate is obtained. Based on these findings we conclude that maturation of mtRNAs is not only dependent on mitochondrial proteins, but also on a more direct interaction of the translation machinery and RNA processing whose nature is so far unknown.

Chloramphenicol↗