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R H Reeder

Publications and source records attributed to R H Reeder.

At least 55 records · Page 3Linked to original sources
PubMed1988

The Xenopus laevis ribosomal gene promoter contains a binding site for nuclear factor-1.

Nuclear Factor I (NF1) is a DNA binding protein that is known to function in the replication of Adeno virus and also binds to many promoters recognized by RNA polymerase II. We have found that there is also an NF1 binding site within the ribosomal gene promoter from Xenopus laevis as well as in several other promoters recognized by RNA polymerase I. The function of a binding site for a polymerase II transcription factor within a promoter recognized by polymerase I is not known. However, its presence suggests interesting regulatory possibilities.

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PubMed1988

Sequence elements essential for function of the Xenopus laevis ribosomal DNA enhancers.

The intergenic spacer region of the Xenopus laevis ribosomal DNA contains multiple elements which are either 60 or 81 base pairs long. Clusters of these elements have previously been shown to act as position- and distance-independent enhancers on an RNA polymerase I promoter when located in cis. By a combination of deletion and linker scanner mutagenesis we show that the sequences essential for enhancer function are located within a 56-base-pair region that is present in both the 60- and 81-base-pair repeats. Within the 56-base-pair region one linker scanner mutation was found to be relatively neutral, suggesting that each enhancer element may be composed of two smaller domains. Each 56-base-pair region appears to be an independent enhancer with multiple enhancers being additive in effect. We review the current evidence concerning the mechanism of action of these enhancers.

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PubMed1987

Linker scanner mutagenesis of the Xenopus laevis ribosomal gene promoter.

We have assayed a series of linker scanner mutants which cover the Xenopus laevis ribosomal gene promoter at approximately ten base pair intervals. All of these mutations adversely affect promoter activity with the exception of one mutation which stimulates activity. Thus, none are neutral. We show that most of the mutations can be partially rescued by ligating a block of enhancer elements upstream of the promoter. In addition, we have made extracts from liver nuclei which produce DNaseI protection footprints over the promoter. Analysis of both strands reveals a prominent footprinting domain from about -5 to -30. However, lesser changes in the digestion pattern are detected over most of the promoter. Previously published analyses have suggested that this promoter might be composed of three functional domains. The experiments presented here suggest that either 1) the three putative domains are so closely arranged that the boundaries are difficult to discern, or 2) the situation is more complex.

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PubMed1987

Ribosomal precursor 3' end formation requires a conserved element upstream of the promoter.

In Xenopus laevis the 3' end of the longest intact ribosomal RNA precursor is formed by a processing event at site T2, which is located 7860 bp downstream of the site of transcription initiation. Processing at T2 is eliminated by mutations within the T2 box, a 7 nucleotide conserved element, GACTTGC, located 15 bp downstream of the 3' ends. The same conserved box is also present at T3, a site 60 bp upstream of the gene promoter and that is part of a termination site. Surprisingly, mutations within the T3 box also eliminate processing at T2. To obtain proper T2 function, T3 can be at any distance but must be in the correct orientation upstream of a ribosomal gene promoter.

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PubMed1987

Heat shock stabilizes highly unstable transcripts of the Xenopus ribosomal gene spacer.

We have shown recently that, in Xenopus laevis oocytes, the 3' end of the longest detectable ribosomal precursor RNA is not formed by transcription termination but by RNA processing and that RNA polymerase I continues to transcribe through the intergenic spacer region. In oocytes, these spacer transcripts are turned over rapidly, and the only apparent transcription termination site is located 215 base pairs upstream of the 5' end of the next transcription unit. In this paper we show that, at heat shock temperature (34 degrees C), processing at the 3' end of the precursor, rapid turnover of spacer transcripts, and termination are all severely impaired. In contrast, transcription initiation and chain elongation are not significantly affected by heat shock. This results in the appearance of large RNA in the range of 10-20 kilobases and longer.

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PubMed1987

A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.

rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.

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PubMed1986

A termination site for Xenopus RNA polymerase I also acts as an element of an adjacent promoter.

On the Xenopus laevis ribosomal genes, RNA polymerase traverses the entire repeating unit of gene plus spacer and terminates just upstream of the next gene promoter. A conserved 7 bp element located at about -200 is an essential part of this terminator. In this paper we show that, in addition to its termination function, this same sequence motif acts as an upstream element of the adjacent promoter and appears to contribute to the long-term stability of the transcription complex.

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PubMed1986

Characterization of three sites of RNA 3' end formation in the Xenopus ribosomal gene spacer.

We have studied three sites of 3' end formation on the Xenopus laevis ribosomal genes, designated T1, T2, and T3. Site T1 coincides with the unique HindIII site at the 3' end of the 28S sequence and is shown to be a site of rapid RNA processing. Transcription continues another 235 bp past T1 to site T2, which marks a boundary between relatively stable and highly unstable transcripts. However, T2 does not cause polymerase release. In nuclear run-off experiments transcription is detected across the entire spacer until site T3, 215 bp upstream of the gene promoter. T3 shares some sequence homology with T2 but appears to cause polymerase release and is presently the best candidate for a true terminator of transcription on these genes.

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PubMed1985

Xenopus ribosomal gene enhancers function when inserted inside the gene they enhance.

The ribosomal DNA of Xenopus laevis contains repeated sequence elements in the intergenic spacer region that enhance transcription from the adjacent gene promoter (1,2). Previous work has shown that these RNA polymerase I enhancers influence the target promoter when they are in either orientation, at a distance of several kilobases, and only when they are in cis (3-5). In this work, we further show that enhancer activity is unaffected by inserting the enhancers within the transcription unit whose promoter is being enhanced. In addition, enhancer activity does not interfere with transcription through its sequences. The results suggest that the enhancers act at a point prior to the initiation of transcription and that they are likely to be dispensable once transcription has begun.

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PubMed1985

DNase I footprinting shows three protected regions in the promoter of the rRNA genes of Xenopus laevis.

Extracts prepared from Xenopus laevis oocytes contain a protein(s) which specifically protects three discrete regions of the RNA polymerase I promoter from digestion by DNase I. Protected region I, from nucleotide +15 to nucleotide -10, spans the site of transcription initiation. Protected region II extends from nucleotide -70 to nucleotide -100 relative to initiation, falling within a 42-base-pair sequence which is homologous to the 60/81-base-pair repeated elements which occur outside of the promoter in the spacer. Protected region III is upstream of region II, from nucleotide -120 to nucleotide -140. All three regions correlate with sequences known from deletion studies to be important for promoter function. Deletion mutants which retain either region I or regions II and III together footprint normally. Deletion of region III, however, reduces but does not eliminate footprinting on region II, suggesting either that one protein binds to both regions or that the proteins which bind to these sites interact with each other.

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PubMed1984

Variations in transcriptional activity of rDNA spacer promoters.

We have compared the DNA sequences of several different examples of the duplicated polymerase I promoters that are found in rDNA spacers of Xenopus laevis. Although different spacers exhibit different amounts of transcription in vivo, this does not seem to be due to DNA sequence differences between spacer promoters. We have found that several different spacer promoters when subcloned and injected into oocytes exhibit similar promoter activities when transcription is assayed by primer extension analysis. Moreover, the activity of these spacer promoters is the same as that of a co-injected gene promoter. The equivalence of spacer promoter activity and gene promoter activity was also found when rDNA plasmids containing intact spacers were injected into oocytes and transcription assayed by primer extension. This is in contrast to (1) the inactivity normally exhibited by the promoters of endogenous spacers in oocytes, (2) the relative inactivity of spacer promoters found when transcription of the same rDNA plasmids is assayed by electron microscopy.

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PubMed1984

Effect of topological constraint on transcription of ribosomal DNA in Xenopus oocytes. Comparison of plasmid and endogenous genes.

We have analyzed the effects of topological constraint on the transcription of both injected ribosomal DNA plasmids and the endogenous ribosomal genes in Xenopus oocytes. Efficient transcription of injected ribosomal gene plasmids requires a covalently closed circular template. Once transcription is initiated on injected plasmids there is a continuous requirement for topological constraint, since subsequent cutting with a restriction endonuclease abolishes transcription. In contrast, both initiation and elongation of transcription on endogenous ribosomal genes are maintained after cutting with restriction endonucleases.

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PubMed1984

In vitro methylation of HpaII sites in Xenopus laevis rDNA does not affect its transcription in oocytes.

The effect of methylation of X. laevis rDNA on its transcription was tested by methylating all of the HpaII sites in a series of rDNA plasmids and injecting the methylated rDNA into X. borealis oocytes. Analysis of RNA transcribed from the injected plasmids showed that methylation of rDNA in vitro does not affect transcription of X. laevis ribosomal genes. This result suggests that despite the observed correlation between demethylation of rDNA and synthesis of ribosomal RNA in X. laevis embryos (1), methylation of rDNA does not regulate transcription of the gene.

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PubMed1984

Anucleolate frog embryos contain ribosomal DNA sequences and a nucleolar antigen.

Previous studies of Xenopus laevis embryos homozygous for the nucleolar deletion mutation have concluded that these embryos contain few, if any, copies of the genes for the 18 S and 28 S ribosomal RNAs. Using hybridization to restriction endonuclease digests of DNA it is found, in fact, that a small amount of ribosomal DNA is still present in such embryos. The ribosomal DNA in these embryos appears to include a few normal repeats together with a variety of unusual fragments containing either spacer or gene sequences. An antibody found in the serum of a scleroderma patient reacts with an antigen localized in the nucleoli of wild-type embryos. In anucleolate embryos this antigen is found in the so-called pseudonucleoli and in many small bodies in the nuclei.

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PubMed1984

Enhancer-like properties of the 60/81 bp elements in the ribosomal gene spacer of Xenopus laevis.

The spacer region of the Xenopus laevis ribosomal gene contains blocks of repetitive sequence elements that are 60 or 81 bp long. These 60/81 bp elements function as enhancer elements for the RNA polymerase I promoter at the 5' end of the gene. An RNA polymerase I promoter adjacent to a block of 60/81 bp elements is always dominant over a promoter on a second plasmid when both are coinjected into oocyte nuclei. If two promoters are placed on the same plasmid containing enhancers, both promoters come under their influence and are codominant. The influence of the enhancers can be transmitted through several kilobases of plasmid sequence, through a potentially active promoter, and is independent of the orientation of the enhancers. The enhancers appear to compete with promoters for the same transcription factor(s); however, the enhancers can only compete when they are on a circular plasmid.

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