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B Sollner-Webb

Publications and source records attributed to B Sollner-Webb.

At least 19 recordsLinked to original sources
PubMed1995

ST-1, a 39-kilodalton protein in Trypanosoma brucei, exhibits a dual affinity for the duplex form of the 29-base-pair subtelomeric repeat and its C-rich strand.

In our attempt to identify telomere region-binding proteins in Trypanosoma brucei, we identified ST-1, a polypeptide with novel features. ST-1 was chromatographically purified from S-100 cell extracts and was renatured from a sodium dodecyl sulfate-protein gel as a 39-kDa polypeptide. It forms a specific complex with the trypanosome telomere repeats of TTAGGG, but more significantly, it shows a higher affinity for the 29-bp subtelomere repeats of T. brucei. These 29-mer boxes are a large tandem series of telomere-derived repeats which separate the simple telomere DNA from middle-repetitive telomere-associated sequences on many chromosomes. ST-1 is the first example of a protein binding within such large repetitive subtelomere elements in trypanosomes or other organisms. ST-1 is also novel in that it has a selective affinity for the C-rich strands of both the subtelomeric 29-mer and the telomere repeats, comparable to that for the duplex form of the respective repeats. All previously described telomere-binding proteins have affinity for only the duplex form or for the G-rich strand. This C-rich strand binding specificity of ST-1 may provide insight into this protein's mechanism of binding in vivo.

Animals↗
PubMed1995

Editing domains of Trypanosoma brucei mitochondrial RNAs identified by secondary structure.

The posttranscriptional insertion and deletion of U residues in trypanosome mitochondrial transcripts called RNA editing initiates at the 3' end of precisely defined editing domains that can be identified independently of the cognate guide RNA. The regions where editing initiates in Trypanosoma brucei cytochrome b and cytochrome oxidase subunit II preedited mRNAs are specifically cleaved by a trypanosome mitochondrial endonuclease that acts like mung bean nuclease and therefore is single strand specific. The regions where editing initiates in virtually all examined preedited mRNAs are predicted to form loop structures, suggesting that editing domains could generally be recognized as prominent single-stranded loops. In contrast to preedited mRNA, edited mRNA can be either resistant or sensitive to cleavage by trypanosome mitochondrial endonuclease, depending on the reaction conditions. This selectivity appears dependent on the availability of extract RNAs, and in model reactions, edited mRNA becomes resistant to cleavage upon base pairing with its guide RNA. Natural partially edited mRNAs are also specifically cleaved with a sensitivity like preedited and unlike edited mRNAs, consistent with their being intermediates in editing. These results suggest that in vivo, the structure of editing domains could initially be recognized by the mitochondrial endonuclease, which could target its associated RNA ligase and terminal U transferase to begin cycles of enzymatic editing modifications.

Animals↗
PubMed1995

Trypanosoma brucei mitochondrial guide RNA-mRNA chimera-forming activity cofractionates with an editing-domain-specific endonuclease and RNA ligase and is mimicked by heterologous nuclease and RNA ligase.

RNA editing in trypanosomes has been proposed to occur through transesterification or endonuclease cleavage and RNA ligation reactions. Both models involve a chimeric intermediate in which a guide RNA (gRNA) is joined through its 3' oligo(U) tail to an editing site of the corresponding mRNA. Velocity centrifugation of Trypanosoma brucei mitochondrial extracts had been reported to completely separate the gRNA-mRNA chimera-forming activity from endonuclease activity (V. W. Pollard, M. E. Harris, and S. L. Hajduk, EMBO J. 11:4429-4438, 1992), appearing to rule out the endonuclease-RNA ligase mechanism. However, we show that an editing-domain-specific endonuclease activity does cosediment with the chimera-forming activity, as does the RNA ligase activity, but detection of the specific endonuclease requires reducing assay conditions. This report further demonstrates that the T. brucei chimera-forming activity is mimicked by mung bean nuclease and T4 RNA ligase. Using cytochrome b (CYb) preedited mRNA and a model CYb gRNA, we found that these heterologous enzymes specifically generate CYb gRNA-mRNA chimeras analogous to those formed in the mitochondrial extract. These combined results provide support for the endonuclease-RNA ligase mechanism of chimera formation.

Animals↗
PubMed1995

Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract.

RNA editing in kinetoplast mitochondrial transcripts involves the insertion and/or deletion of uridine residues and is directed by guide RNAs (gRNAs). It is thought to occur through a chimeric intermediate in which the 3' oligo(U) tail of the gRNA is covalently joined to the 3' portion of the mRNA at the site being edited. Chimeras have been proposed to be formed by a transesterification reaction but could also be formed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract. This reaction was found to occur in two steps. First, the mRNA is cleaved in the 3' portion of the editing domain, and then the 3' fragment derived from this cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage product is a more efficient substrate for chimera formation than is the intact mRNA, inconsistent with a transesterification mechanism but supporting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera formation no longer occurs. Rather, this phosphorothioate induces both cleavage and chimera formation at a novel site within the editing domain. Finally, levels of chimera-forming activity correlate with levels of mitochondrial RNA ligase activity when reactions are conducted under conditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation in the mitochondrial extract occurs by a nuclease-ligase mechanism rather than by transesterification.

Animals↗
PubMed1995

Stimulation of the mouse rRNA gene promoter by a distal spacer promoter.

We show that the mouse ribosomal DNA (rDNA) spacer promoter acts in vivo to stimulate transcription from a downstream rRNA gene promoter. This augmentation of mammalian RNA polymerase I transcription is observed in transient-transfection experiments with three different rodent cell lines, under noncompetitive as well as competitive transcription conditions, over a wide range of template concentrations, whether or not the enhancer repeats alone stimulate or repress expression from the downstream gene promoter. Stimulation of gene promoter transcription by the spacer promoter requires the rDNA enhancer sequences to be present between the spacer promoter and gene promoter and to be oriented as in native rDNA. Stimulation also requires that the spacer promoter be oriented toward the enhancer and gene promoter. However, stimulation does not correlate with transcription from the spacer promoter because the level of stimulation is not altered by either insertion of a functional mouse RNA polymerase I transcriptional terminator between the spacer promoter and enhancer or replacement with a much more active heterologous polymerase I promoter. Further analysis with a series of mutated spacer promoters indicates that the stimulatory activity does not reside in the major promoter domains but requires the central region of the promoter that has been correlated with enhancer responsiveness in vivo.

Animals↗
PubMed1992

Specific cleavage of pre-edited mRNAs in trypanosome mitochondrial extracts.

RNA editing in Trypanosoma brucei is a posttranscriptional processing event that results in the addition and deletion of uridine residues within several mitochondrial mRNAs. We have examined reactions involving pre-edited precursor RNAs in vitro. In this study, we report specific cleavage of pre-edited cytochrome b (CYb), cytochrome oxidase subunit II (COII), and cytochrome oxidase subunit III (COIII) mRNAs when incubated with T. brucei mitochondrial extracts. The pre-edited CYb RNA was cleaved near the 3'-most uridine addition sites, within the region where editing would be expected to commence. Pre-edited COII mRNA was similarly cleaved adjacent to its small editing domain, while pre-edited COIII RNA was cleaved at multiple sites in the region where uridine addition and deletion occurs in vivo. In contrast, edited versions of CYb, COII, and COIII RNAs were not cleaved within the editing domains. Such differential cleavage of the edited and pre-edited forms of these mRNAs suggests either a direct involvement in RNA editing or involvement in another aspect of mitochondrial gene expression requiring cleavage of pre-edited RNAs.

Animals↗
PubMed1991

Stable integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination.

A calmodulin-neomycin-resistance fusion gene was introduced into Trypanosoma brucei by electroporation, and stably transformed cell lines were obtained. In all of the transformants, the fusion gene had integrated into the host genome at the cognate locus, evidently by homologous recombination within flanking calmodulin DNA. This unusual observation distinguishes trypanosomes as the only eukaryote other than yeast known to undergo gene targeting in essentially 100% of the stable transformants. It should now be possible to systematically manipulate the trypanosome genome, directing predetermined mutations to virtually any chromosomal locus.

Animals↗
PubMed1991

RNA editing.

Since its discovery, RNA editing in kinetoplastid mitochondria has proven a fascinating topic of study, and the last one and a half years have witnessed enormous advances in our understanding of this unprecedented form of RNA processing. The information flow in this RNA editing, once considered a candidate for defying the central dogma, is now known to conform to the DNA-to-RNA-to-protein paradigm, with the novel feature that the sequence of an edited region is not actually present in any DNA segment, but instead derives by a novel micro-interdigitating of information encoded in multiple DNA regions.

Mitochondria↗
PubMed1991

News from the nucleolus: rRNA gene expression.

Although the typical, actively growing eukaryotic cell contains over 10,000 different transcripts, half of its RNA synthetic capacity is devoted to the production of a single kind of RNA. This is the pre-ribosomal RNA, which is synthesized in a special compartment of the nucleus, the nucleolus, and is the exclusive product of transcription by RNA polymerase I. In vivo and in vitro approaches have revealed the major features of rRNA gene transcription and of the subsequent processing of the primary transcript.

Animals↗
PubMed1991

Homologous recombination in the tandem calmodulin genes of Trypanosoma brucei yields multiple products: compensation for deleterious deletions by gene amplification.

Homologous recombination between a calmodulin-neomycin-resistance fusion gene and the Trypanosoma brucei chromosome takes place not only in the large 5'- and 3'-flanking segments of the calmodulin locus but also in any of the four tandem genomic calmodulin genes. This results in a recombined locus consisting of the chimeric neor gene and four, three, two, one, or zero functional calmodulin genes. Cells bearing this latter event have half of their normal number of intact calmodulin genes and an accompanying phenotype of slow growth. Over months of propagation, these lines acquire additional calmodulin genes, frequently by amplifying a calmodulin gene at the untargeted locus, and concomitantly revert to normal growth rate. This response could be related to the property of the trypanosome of maintaining most housekeeping genes in tandem chromosomal arrays. Recombination appears to be initiated by a crossover event between the linearized end of the transfecting plasmid and a homologous region in the host genome; the second crossover generally occurs internally and in that region requires no more than 87 bp of homology.

Animals↗
PubMed1991

Sequence organization and RNA structural motifs directing the mouse primary rRNA-processing event.

The first processing step in the maturation of mouse precursor rRNA involves cleavage at nucleotide ca. +650, at the 5' border of a 200-nucleotide region that is conserved across mammals and contains the sequences that direct the processing. To identify the relevant sequence elements, we used rRNAs with small internal mutations and short pre-rRNA substrates. Much of the region can be mutated without appreciable effect, but nucleotides +655 to +666 appear to be absolutely required and short segments surrounding +750 and +810 markedly stimulate processing. The minimal processing signal corresponds to rRNA nucleotides +645 to +672. Formation of a ribonucleoprotein complex of retarded electrophoretic mobility is evidently necessary but not sufficient for processing. Computer-assisted analysis suggested a phylogenetic- and mutant-supported secondary structure in which the minimal processing signal forms a stem with the +655 region in the loop, and there is a separate branched duplex containing the downstream stimulatory sequences. Use of antisense RNA, in trans and in cis, to sequester the +655 region in a duplex supported the hypothesis that this critical region was needed in a single-stranded conformation for processing and for specific complex formation.

Animals↗
PubMed1990

RNA editing involves indiscriminate U changes throughout precisely defined editing domains.

RNA editing is the specific posttranscriptional insertion/deletion of U residues within trypanosomatid mitochondrial transcripts. We used a novel cDNA cloning scheme and analyzed partially edited COIII and CYb RNAs. Our major unanticipated findings are: First, editing appears strikingly indiscriminate within editing domains, inserting and deleting variable numbers of U residues at all sites, both those requiring editing and those that do not. Second, this incomplete editing occurs in precise domains, not in adjacent regions unedited in mature mRNA. Third, editing appears to commence prior to polyadenylation. We propose that within a region being edited, there are cycles of indiscriminate cleavage, U addition or deletion, and religation; sites that become correctly edited would be protected from further modification by duplexing with short RNAs complementary to the edited sequence.

Animals↗
PubMed1990

The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing.

The first cleavage in mammalian pre-rRNA maturation occurs near the 5' end within the 5' external transcribed spacer. Using mouse cell extracts, we show that this processing is abolished by micrococcal nuclease pretreatment. Autoantibodies that recognize the U3, U8, and U13 snRNPs (anti-fibrillarin) deplete processing activity from the extract and selectively immunoprecipitate both rRNA substrates and processing products from the reaction. Specific involvement of the U3 snRNP is demonstrated by native gel electrophoresis of the processing reaction followed by Northern blotting and by oligonucleotide-directed RNAase H abolition of processing activity. Our identification of U3 function is discussed with respect to the molecular basis of pre-rRNA recognition by the U3 snRNP, possible roles of U3 and other nucleolar snRNPs in rRNA processing, and the morphological organization of the nucleolus and the ribosomal transcription complex.

Animals↗
PubMed1990

Half helical turn spacing changes convert a frog into a mouse rDNA promoter: a distant upstream domain determines the helix face of the initiation site.

Transcription of frog rDNA by mouse cell factors is the only documented exception to the observed species selectivity of rRNA gene expression. This heterologous transcription is authentic in that it uses the normal frog upstream and core promoter domains, as well as the normal mouse polymerase I transcription factors, but it initiates at residue -4. We now show that by introducing an insertion or deletion of approximately one-half helical turn anywhere within the 90-bp region between the upstream and core promoter domains, the initiation site moves to residue +1. Promoters bearing spacing changes of approximately one or two full helix turns do not initiate at residue +1, whereas a promoter with a one and one-half-turn helical turn spacing change again supports initiation at residue +1. Thus, the position of the upstream domain of the frog promoter shows a stereo-specific requirement relative to the core promoter domain and dictates the face of the DNA helix on which transcription initiates, 140 bp away. In contrast, relative to the core promoter domain, initiation can occur on either side of the DNA helix. Furthermore, the striking observation that several frog half helical turn spacing change mutants are stronger templates with the mouse factors than the homologous mouse rDNA suggests that the polymerase I transcriptional machinery of even distantly related species is far more similar than generally envisioned.

Animals↗
PubMed1990

A rapidly rearranging retrotransposon within the miniexon gene locus of Crithidia fasciculata.

The tandemly arrayed miniexon genes of the trypanosomatid Crithidia fasciculata are interrupted at specific sites by multiple copies of an inserted element. The element, termed Crithidia retrotransposable element 1 (CRE1), is flanked by 29-base-pair target site duplications and contains a long 3'-terminal poly(dA) stretch. A single 1,140-codon reading frame is similar in sequence to the integrase and reverse transcriptase regions of retroviral pol polyproteins. Cloned lines derived from a stock of C. fasciculata have unique arrangements of CRE1s. In different cloned lines, CRE1s, in association with miniexon genes, are located on multiple chromosomes. By examining the arrangement of CRE1s in subclones, we estimate that the element rearranges at a rate of ca. 1% per generation. These results indicate that the C. fasciculata miniexon locus is the target for a novel retrotransposon.

Amino Acid Sequence↗
PubMed1990

Enhancers for RNA polymerase I in mouse ribosomal DNA.

The intergenic spacer of the mouse ribosomal genes contains repetitive 140-base-pair (bp) elements which we show are enhancers for RNA polymerase I transcription analogous to the 60/81-bp repetitive enhancers (enhancers containing a 60-bp and an 81-bp element) previously characterized from Xenopus laevis. In rodent cell transfection assays, the 140-bp repeats stimulated an adjacent mouse polymerase I promoter when located in cis and competed with it when located in trans. Remarkably, in frog oocyte injection assays, the 140-bp repeats enhanced a frog ribosomal gene promoter as strongly as did the homologous 60/81-bp repeats. Mouse 140-bp repeats also competed against frog promoters in trans. The 140-bp repeats bound UBF, a DNA-binding protein we have purified from mouse extracts that is the mouse homolog of polymerase I transcription factors previously isolated from frogs and humans. The DNA-binding properties of UBF are conserved from the mouse to the frog. The same regulatory elements (terminators, gene and spacer promoters, and enhancers) have now been identified in both a mammalian and an amphibian spacer, and they are found in the same relative order. Therefore, this arrangement of elements probably is widespread in nature and has important functional consequences.

Animals↗