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B Sollner-Webb

Publications and source records attributed to B Sollner-Webb.

At least 73 records · Page 4Linked to original sources
PubMed1987

Primary processing of mammalian rRNA involves two adjacent cleavages and is not species specific.

The primary transcript of the mouse rRNA gene is rapidly processed at nucleotide approximately +650 both in vivo and in vitro. Using run-off transcription in a mouse cell extract as well as S1 nuclease and primer extension analysis of cellular RNA, we demonstrated that this primary processing actually results in the formation of two species of downstream RNA which differ in length by approximately 6 nucleotides, indicating the existence of two closely positioned alternative processing sites. The 200-base-pair region just 3' to the mouse processing site has a striking 80% sequence homology with a region of the human rRNA external transcribed spacer, and S1 nuclease analysis of human cellular RNA has demonstrated that an analogous rRNA processing occurs at the 5' border of the homologous human region. Unlike rDNA transcriptional initiation, however, the primary rRNA processing is not highly species specific, for the transcript of a chimeric gene containing the human processing region adjacent to a mouse rDNA promoter was synthesized and correctly processed in a mouse cell extract. This result confirms that mouse and human rRNA undergo a common primary processing event which is evidently directed by sequences within the 200-base-pair conserved sequence region.

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PubMed1986

A transcriptional terminator is a novel element of the promoter of the mouse ribosomal RNA gene.

Sequences flanking residue-168 of the mouse rRNA gene are essential to direct efficient transcription in transfected cells and are stimulatory in vitro on closed circular templates. This promoter domain evidently functions by the unprecedented mechanism of terminating polymerase I-directed transcripts. It inhibits transcripts from reading into the initiation region, acting cotranscriptionally to end these RNAs at residue--182 and release them from the template. Most likely, polymerases on tandem genomic rRNA genes are not released upon completing each 40-47S transcript, but traverse the entire spacer to the next promoter-terminator, where they are made available and positioned to favor reinitiation. Through such polymerase recycling, plus the binding of free polymerase, the rDNA promoters could achieve their characteristically high level of transcription.

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PubMed1986

RNA polymerase specificity of mRNA production and enhancer action.

To examine the RNA polymerase (EC 2.7.7.6) specificity of RNA maturation/utilization and transcriptional enhancement, we constructed a chimeric plasmid (pPolI-CAT) in which a promoter for mouse rRNA gene transcription was placed adjacent the coding sequences for chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). A number of other constructs, including plasmids also containing a murine sarcoma virus enhancer or lacking any natural eukaryotic promoter sequences, were also prepared. In apparent agreement with earlier conclusions that an RNA polymerase I transcript can act as a messenger RNA, transient transfection of mouse L cells with pPolI-CAT yielded both high levels of transcription from the RNA polymerase I promoter and enzymatically active CAT protein. However, further examination revealed that CAT protein is not translated from RNA that begins at the normal rRNA transcription initiation site. Polysomal RNA is devoid of such RNA and instead consists of CAT-encoding transcripts that begin elsewhere in the mouse ribosomal DNA (rDNA) region. Since transcription of these aberrant RNAs is stimulated by the addition of a murine sarcoma virus enhancer segment, they are probably transcribed by RNA polymerase II. Transcripts that map to the authentic rRNA start site are not similarly enhanced. Moreover, unlike the RNAs deriving from the rRNA initiation site, these aberrant RNAs are more stable and the level of translatable CAT transcripts is suppressed by inclusion of larger segments of the rDNA promoter regions. Fortuitously initiated mRNAs are also formed in the absence of any natural eukaryotic promoter sequence. From these data we conclude that there is no evidence that normal RNA polymerase I transcription yields functional mRNA and that transcriptional enhancement appears to be RNA polymerase specific.

Acetyltransferases↗
PubMed1986

Factors and nucleotide sequences that direct ribosomal DNA transcription and their relationship to the stable transcription complex.

We have studied the protein components and nucleic acid sequences involved in stably activating the ribosomal DNA (rDNA) template and in directing accurate transcription of mammalian rRNA genes. Two protein components are necessary to catalyze rDNA transcription, and these have been extensively purified. The first, factor D, can stably associate by itself with the rDNA promoter region and is responsible for template commitment. The second component, factor C, which appears to be an activated subset of polymerase I, can stably bind to the factor D-rDNA complex but not to the rDNA in the absence of factor D. A third component which had been previously identified as a rDNA transcription factor is shown to be a RNase inhibitor. Extending our earlier observation that the approximately 150-base-pair mouse rDNA promoter consists of a minimal essential region (residues approximately -35 to approximately +9) and additional upstream stimulatory domains, we now report that each of these promoter domains acts to augment the binding of the polymerase I transcription factors. A minimum core region (residues approximately -35 to approximately -15) is capable of stable complex formation and of binding transcription factor D. Factor C can also bind to this D-core region complex.

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PubMed1986

Two distant and precisely positioned domains promote transcription of Xenopus laevis rRNA genes: analysis with linker-scanning mutants.

To examine the internal organization of the promoter of the Xenopus laevis rRNA gene, we constructed a series of linker-scanning mutants that traverse the rDNA initiation region. The mutant genes, which have 3 to 11 clustered base substitutions set within an otherwise unaltered rDNA promoter sequence, were injected into Xenopus oocyte nuclei, and their transcriptional capacity was assessed by S1 nuclease analysis of the resultant RNA. The data demonstrate that there are two essential promoter domains, the distal boundaries of which coincide with the promoter boundaries established previously by analysis of 5' and 3' deletion mutants. The upstream promoter domain is relatively small and extends from residues ca. -140 to -128. The downstream domain is considerably larger, encompassing residues ca. -36 to +10, and exactly corresponds in both size and position to the mammalian minimal promoter region. The Xenopus rDNA sequence between these two essential domains has a much smaller effect on the level of transcriptional initiation. In light of the fact that a large portion of this intervening region consists of a segment (residues -114 to -72) that is duplicated many times in the upstream spacer to form an rDNA enhancer sequence, it is noteworthy that a "-115/-77 linker scanner," in which virtually this entire segment is replaced by a polylinker sequence, has full promoter activity in the injected Xenopus borealis oocytes. Analysis of a parallel series of spacing change linker-scanning mutants revealed the unexpected result that the relative positions of the upstream and downstream promoter domains are very critical: all spacing alterations of more than 2 base pairs within this 100-base-pair region virtually abolish promoter activity. We conclude that the factors that bind to these two distant promoter domains must interact in a very precise stereospecific manner.

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PubMed1986

Upstream domains of the Xenopus laevis rDNA promoter are revealed in microinjected oocytes.

The DNA sequences involved in promoting transcription of the Xenopus laevis rRNA genes were determined by microinjecting a series of deletion mutants into oocyte nuclei. A very small promoter region is sufficient to direct efficient transcription when templates are microinjected at high rDNA concentration, since 5'delta- 9 and 3'delta +6 templates are fully active. However, as the concentration of injected template is decreased, an increasing requirement for upstream domains, extending to nucleotide approximately -170, is observed. The major downstream border of the required region does not change. This apparently expanding 5' promoter border results from the fact that, as the rDNA concentration is decreased, transcription from templates lacking the upstream promoter domain falls off much more sharply than does transcription from a complete promoter. In fact, the deleted promoters are virtually inactive below a threshold rDNA concentration. It is indeed the rDNA concentration that is important, for coinjected vector DNA does not increase the level of transcription obtained from low concentrations of the 5' deletions. From these data we conclude that polymerase I transcription factors can recognize and initiate transcription from a small core promoter domain, but that sequences extending upstream to nucleotide approximately -170 increase the efficiency of initiation. A model is presented that could account for these results.

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PubMed1985

Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses.

Although the technique of S1 mapping is a powerful analytical tool for the analysis of RNA, we now report a surprising complication involving a trimolecular hybrid between two RNA species and a single DNA probe molecule which, if unrecognized, can lead to misleading interpretations. We document that such trimolecular hybrids can be efficiently formed under some hybridization conditions and that the probe DNA sequence at the junction of the two RNA molecules can be remarkably stable to digestion with S1. Trimolecular hybrids can arise in any instance whenever a distal region of an end-labeled DNA probe is homologous to a moderately abundant RNA in the sample to be analyzed. This situation presents a serious, potential complication for a variety of S1 analyses, particularly those in which DNA transfection has been utilized to reintroduce in vitro-engineered genes into cultured animal cells.

Acetyltransferases↗
PubMed1985

A complex control region of the mouse rRNA gene directs accurate initiation by RNA polymerase I.

To determine the size and location of the mouse rDNA promoter, we constructed systematic series of deletion mutants approaching the initiation site from the 5' and 3' directions. These templates were transcribed in vitro under various conditions with S-100 and whole-cell extracts. Surprisingly, the size of the rDNA region that determines the level of transcription differed markedly, depending on the reaction conditions. In both kinds of cell extracts, the apparent 5' border of the promoter was at residue ca. -27 under optimal transcription conditions, but as reaction conditions became less favorable, the 5' border moved progressively out to residues -35, -39, and -45. The complete promoter, however, extends considerably further, for under other nonoptimal conditions, we observed major effects of promoter domains extending in the 5' direction to positions ca. -100 and -140. In contrast, the apparent 3' border of the mouse rDNA promoter was at residue ca. +9 under all conditions examined. We also show that the subcloned rDNA region from -39 to +9 contains sufficient information to initiate accurately and that the region between +2 and +9 can influence the specificity of initiation. These data indicate that, although the polymerase I transcription factors recognize and accurately initiate with only the sequences downstream of residue -40, sequences extending out to residue -140 greatly favor the initiation reaction; presumably, this entire region is involved in rRNA transcription in vivo.

Animals↗
PubMed1985

Eucaryotic transcription complexes are specifically associated in large sedimentable structures: rapid isolation of polymerase I, II, and III transcription factors.

RNA synthesis in eucaryotes takes place on template molecules that are activated by stably associating with limiting transcription factors. In this paper we demonstrate that such stable transcription complexes can be specifically sedimented from in vitro transcription reaction mixtures by mild centrifugation. This occurs with stable complexes of genes transcribed by all three classes of eucaryotic RNA polymerase and with S-100 as well as whole-cell extracts. However, the transcriptional capacity of the isolated complex differs for the three polymerase classes. The pelleted ribosomal DNA (polymerase I) complex contains all the factors necessary for transcription, each purified 25- to 50-fold, whereas the pelleted adenovirus major late promoter (polymerase II) complex lacks a factor that remains in the supernatant. In the case of 5S DNA (polymerase III), a necessary factor associates slowly with the sedimentable complex. Notably, the interactions responsible for this rapid sedimentation are specific for DNA molecules in stable complexes, suggesting that the in vitro sedimentable complex mirrors the in vivo structural organization of active genes.

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PubMed1984

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.

Acetyltransferases↗
PubMed1984

The absence of a human-specific ribosomal DNA transcription factor leads to nucleolar dominance in mouse greater than human hybrid cells.

The basis for nucleolar dominance in mouse-human cell hybrids which contained all of the mouse chromosomes but an incomplete set of human chromosomes (M greater than H) was examined at the molecular level. S1 mapping data showed that these cells had the expected levels of steady-state rRNA transcribed from mouse ribosomal gene (rDNA) transcription units but undetectable levels of rRNA derived from the human rDNA transcription templates that are also present. RNA polymerase I-dependent, cell-free transcription extracts were made from three hybrid lines and were found to be capable of transcribing cloned rDNA templates of mouse but not human origin. Partially purified human factors required for rDNA transcription in vitro were added to the M greater than H extracts. One fraction with almost no RNA polymerase I activity conferred on these hybrid cell extracts the ability to transcribe a human rDNA template. These rescue experiments suggested that this required human-specific rDNA transcription factor(s) was effectively absent from the lines we examined and could account for nucleolar dominance in M greater than H hybrid cells.

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PubMed1983

Dinucleotide primers facilitate convenient identification of the mouse ribosomal DNA transcription initiation site. A general method for analysis of transcription by RNA polymerases I and III.

The in vitro initiation site for RNA polymerase I on the mouse rRNA gene was identified using a new method that is generally applicable to the study of other eukaryotic transcripts. First, the 5' end of mouse rRNA was located to an ApC . . . by high resolution S1 nuclease mapping. Dinucleotide primers were then used in transcription reactions to demonstrate that this position is the actual de novo initiation site, and not a rapid RNA processing site. For this analysis, initiation was inhibited by reduced rXTP concentration, and, upon supplementation with various dinucleotides, only ApC stimulated correct synthesis. Independently confirming its role as the initiating nucleotide, ATP was shown to be required at a much higher concentration than the other rXTPs for RNA initiation, but not for elongation. These results also demonstrate a marked sequence conservation of rRNA initiation sites between the mouse and frog, two species that violate the general rule of species specificity in RNA polymerase I initiation. Extending these studies to RNA polymerase III, the initiation site for 5 S RNA can be similarly located by dinucleotide analysis and confirmed from the concentration requirements of each rXTP. In addition to allowing initiation at suboptimal rXTP concentration, dinucleotide primers can also circumvent the need for a factor normally required for initiation, suggesting their potential value in dissecting the mechanism of eukaryotic transcription.

Animals↗
PubMed1983

A stable transcription complex directs mouse ribosomal RNA synthesis by RNA polymerase I.

Ribosomal RNA is synthesized from template molecules that are activated by a stable association with essential transcription factors. This activated template assembles prior to the onset of transcription as a preinitiation complex and factors remain firmly attached during active elongation as well. Sequential addition of differently marked rRNA genes to an S-100 mouse cell extract shows that the DNA binding factors of the stable complex are present in limiting quantities. They associate rapidly with template molecules and the resultant transcription complex remains intact over prolonged periods of incubation in the presence of competitor DNA. The resistance of the stable complex to the usual inhibitory effect of high DNA concentration suggests that more than one DNA binding factor recognizes the rDNA promoter region and is needed to direct faithful transcription. Finally, although the stable complex is specific for the rRNA initiation region, added vector sequences can neutralize nonspecific DNA binding components that are also present in the cell extract. This lowers the requirement for rDNA template and demonstrates that each activated rRNA gene can direct at least 10 rounds of elongation and reinitiation.

Animals↗
PubMed1983

Nested control regions promote Xenopus ribosomal RNA synthesis by RNA polymerase I.

The DNA regions required for accurate initiation by RNA polymerase 1 on the ribosomal RNA genes of Xenopus laevis consist of at least two domains within a 150 bp segment adjoining the initiation site. A region of only 13 nucleotides (from -7 to +6) appears sufficient to specify accurate and efficient initiation when various 5' and 3' deletion mutants are injected into oocyte nuclei. In contrast, a 10-fold larger region (from -142 to +6) is required for maximal synthesis when deletion mutants are assayed in oocyte nuclear homogenates; only a low level of synthesis is specified by the 13 bp promoter domain in this in vitro system. Site-specific mutants demonstrate that a sequence adjoining nucleotide -75 is also essential for efficient in vitro initiation. Under other conditions, transcription can be strongly affected by sequences that extend several kb upstream in the rDNA spacer and contain duplications of sequences found within the 150 bp promoter. We propose a model in which transcription of the X. laevis rRNA genes is regulated by interaction of at least three sequence domains that extend over a several kb region.

Animals↗
PubMed1982

Transcription of Xenopus ribosomal RNA genes by RNA polymerase I in vitro.

We have developed an in vitro system in which RNA polymerase I accurately transcribes Xenopus ribosomal RNA (rRNA) genes. Cloned Xenopus laevis ribosomal DNA (rDNA) is incubated in a homogenate of manually isolated Xenopus borealis oocyte nuclei. The resultant RNA is analyzed by an S1 nuclease mapping procedure specific for the initiation region of X. laevis rRNA. We show that transcription initiates in vitro on the cloned X. laevis rDNA at the in vivo initiation site. In addition, the dinucleotide ApG significantly stimulates transcription in vitro by acting as a sequence-specific primer. The ApG stimulation is dependent on the template concentration and independent of the ribonucleoside triphosphate levels suggesting that the primer circumvents or augments an initiation-specific factor. We have also investigated transcription of Xenopus rDNA in a mouse cell extract. This heterologous extract can catalyze accurate initiation at a low level. However, under different ionic conditions, this extract transcribes X. laevis rDNA approximately 100-fold more efficiently, but synthesis initiates 4 nucleotides upstream from the in vivo initiation site. There is strong sequence conservation between this start site, the in vivo X. laevis start site, and the in vivo mouse start site suggesting that these sequences are important in RNA polymerase I initiation and that species specificity for RNA polymerase I transcription may not be as great as suggested earlier.

Animals↗
PubMed1982

Accurate transcription of cloned Xenopus rRNA genes by RNA polymerase I: demonstration by S1 nuclease mapping.

We have demonstrated faithful transcriptional initiation of cloned Xenopus rRNA genes upon injection into Xenopus oocytes. This observation has been made possible by the use of an S1 nuclease assay that is both sensitive and quantitative. In order to detect rRNA synthesis from the injected template above the large background of rRNA endogenously present in oocytes, the divergence of ribosomal DNA sequences between two Xenopus species was utilized. Cloned X. laevis ribosomal DNA was injected into the nuclei of X. borealis oocytes. Total oocyte RNA was then isolated and hybridized to a radioactive DNA probe that over laps the 5' end of X. laevis rRNA; endogenous rRNA of the X. borealis oocytes does not hybridize to the probe. RNA/DNA hybrids were treated with S1 nuclease and protected fragments were sized by polyacrylamide gel electrophoresis. RNA made from the injected rDNA protects the same region of probe as does authentic X. laevis precursor rRNA. Thus, transcription appears to initiate on the cloned, microinjected X. laevis rDNA at the same site as is used in vivo. This synthesis is not impaired by coinjection of an amount of alpha-amanitin sufficient to inhibit RNA polymerase II and III; therefore the reaction is mediated by RNA polymerase I. The amount of transcription may be reproducibly quantitated and we have varied a number of parameters in order to maximize transcriptional expression of the injected rDNA. Eight independently isolated X. laevis rDNA clones as well as several subcloned initiation regions of these genes are all accurately transcribed at approximately equal efficiency. This assay should facilitate analysis of several aspects of rRNA transcription, including deleniation of the Xenopus RNA polymerase I promoter location.

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