Search PubMed⌕ Search

Biomedical subjects

B Sollner-Webb

Publications and source records attributed to B Sollner-Webb.

86 records · Page 5Linked to original sources
PubMed1982

Mapping of transcription initiation and termination signals on Xenopus laevis ribosomal DNA.

We have injected cloned derivatives of Xenopus laevis ribosomal genes into X. laevis oocyte nuclei and examined the resulting transcription complexes in the electron microscope. From this work we conclude that the promoter lies somewhere within a region between -320 nucleotides upstream and +113 nucleotides downstream from the site of transcription initiation. This assignment agrees with inferences based on sequence conservation. It further suggests that the duplicated initiation region sequences located further out in the spacer ("Bam islands") are not required for the normal high densities of RNA polymerase loading seen on ribosomal genes. Concerning termination, the cluster of four Ts that forms part of the HindIII restriction site at the 3' end of the gene appears to be part of the normal termination signal. Termination still occurs when only three Ts are present, but reduction to two Ts damages termination. Because clusters of three Ts appear at several sites within the gene, it is likely that sequences adjacent to the T cluster also are required for normal termination. In addition, we present evidence for a fail-safe termination site just upstream from the site of transcription initiation.

Animals↗
PubMed1979

The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis.

In this study, we have located the sites of transcription initiation and termination on a cloned fragment of ribosomal DNA from X. laevis, and have sequenced the surrounding nucleotides. As reported previously (Reeder, Sollner-Webb and Wahn, 1977), about 25% of the 40S rRNA precursor molecules isolated from oocytes have polyphosphate 5' termini and are therefore presumed to represent primary transcripts. These ends hybridize specifically to the 221 bp DNA fragment and removed the overhanging DNA region with S1 nuclease. In the other, we hybridized 40S RNA to a 221 bp fragment of ribosomal DNA. The nucleotides encoding the 5' end of the 40S RNA were located more precisely by two methods. In one, we hybridized 40S RNA to the 221 bp DNA fragment and removed the overhanging DNA region with S1 nuclease. In the other, we hybridized 40S RNA to a smaller DNA fragment and extended the recessed 3' terminus of the DNA using reverse transcriptase. The resultant DNA fragments were sized on sequencing gels. Both determinations map the 5' end of 40S RNA at the same site in the rDNA, about 2250 bp upstream from the Eco RI site in the 18S rRNA coding sequence. At this site we find a DNA sequence beginning AGGGGAAGAC.... which agrees with partial sequence data from the 5' end of polyphosphorylated and bulk 40S rRNA. Features of this region of the ribosomal DNA will be discussed in this paper. A 227 nucleotide region surrounding the initiation site was also sequenced from an independently derived clone and found to differ in only one nucleotide. In addition, a sequence is found about 1100 nucleotides upstream from the 5' end of the gene that has 90% homology to the sequence from nucleotides minus 125 to +4 in the initiation region. At the termination region, X. laevis ribosomal DNA has a single recognition site for the restriction enzyme Hind III in each repeating unit. Using the S1 nuclease technique, the 3' termini of both the 40S precursor and mature 28S rRNA are seen to map within this recognition sequence. The sequence surrounding the Hind III site has striking homology to termination sites recognized by other RNA polymerase classes. Sequences with similar features are also found upstream from the initiation site.

Animals↗
PubMed1978

DNAase I, DNAase II and staphylococcal nuclease cut at different, yet symmetrically located, sites in the nucleosome core.

We have determined the relative location of pancreatic DNAase (DNAase I), spleen acid DNAase (DNAase II) and staphylococcal nuclease cleavage sites in the nucleosome core. Each of these three enzymes cleaves the DNA of chromatin at 10.n nucleotide intervals (n integer); this specificity presumably reflects the internal structure of the nucleosome. We have already reported that DNAase I cleaves nucleosomal DNA so that nearest adjacent cuts on opposite strands are staggered by 2 nucleotides, 3' end extending (Sollner-Webb and Felsenfeld, 1977). Here we show that the nearest cuts made by DNAase II in nucleosomal DNA are staggered by 4 nucleotides, 3' end extending, while cuts made by staphylococcal nuclease have a stagger of 2 nucleotides, 5' end extending. The cutting sites of the three enzymes thus do not coincide. Each pair of staggered cuts, however, is symmetrically located about a common axis-that is, the "dyad axes" that bisect nearest pairs of cutting sites coincide for all three enzymes. This result is consistent with the presence of a true dyad axis in the nucleosome core. Our results support the conclusion that a structural feature of the nucleosome, having a 10 nucleotide periodicity, is the common recognition site for all three nucleases. The position of the cut is determined, however, by the individual characteristics of each enzyme. Sites potentially available to nuclease cleavage span a region of 4 nucleotides out of this 10 nucleotide repeat, and a large fraction of these sites are actually cut. Thus much of the nucleosomal DNA must in some sense be accessible to the environment.

Animals↗
PubMed1977

Pancreatic DNAase cleavage sites in nuclei.

The DNA of nuclei is cleaved by a variety of nucleases in such a way that the cuts on a given strand are always separated by an integral multiple of 10 nucleotides. However, the spacing between cutting sites on opposite strands is not known for any nuclease. In this paper, we describe the determination of the spacing, or stagger, between cuts on opposite strands produced by the action of pancreatic DNAase (DNAase I) on nuclei. When nuclei are digested with DNAase I and the resultant DNA is analyzed by gel electrophoresis without prior denaturation, a complex pattern of bands is observed. A method which gives better than 90% recovery of DNA from polyacrylamide gels was used to isolate the individual fractions corresponding to these bands. The structure of the fractions was then determined using single-strand-specific nuclease to digest single-stranded "tails" and using DNA polymerases to extend recessed 3'-OH termini of partially duplex regions. Our results show that each component consists of a double-stranded region terminating in single-stranded tails at both ends. Although both chains of every duplex are 10-n nucleotides long (n integer), the chains are never completely paired. The experiments with DNA polymerase show an abundance of structures in which the 3'-OH termini of these duplexes are recessed by 8 nucleotides, and by inference, there must be structures with 5'-P termini recessed by 2 or 12 nucleotides. Thus DNAase I acts on nuclei to produce DNA with staggered cuts on opposite strands, separated by (10-n + 8) and (10-n + 2) base pairs (with 5'-P and 3'-OH termini extending, respectively). Two classes of models of DNA folding in the nucleosome have been proposed by other investigators to account for the presence of DNAase I cleavage sites at 10-n intervals along each DNA chain. One class of models leads to the prediction that cuts should either be unstaggered or separated by 10 nucleotides, while the other class is consistent with staggers of 6 and 4 nucleotides. Neither prediction is verified by our data; however, all these models may be made consistent with the results by assuming that the enzyme's site of recognition on nucleosomal DNA is not the same as its site of cleavage.

Animals↗
PubMed1977

Sites of transcription initiation in vivo on Xenopus laevis ribosomal DNA.

We report the results of a novel method for locating sites of transcription initiation using a complex of capping enzymes from vaccinia virions that catalyze the reaction pppG + S-adenosylmethionine + (p)ppXpYpZp..... -->(7m)GpppXpYpZp..... [Ensinger, M. J., Martin, S. A., Paoletti, E. and Moss, B. (1975) Proc. Natl. Acad. Sci. USA 72, 2525-2529]. This enzyme complex will cap di- or triphosphate termini but will not cap monophosphate or hydroxyl termini. Xenopus laevis 40S precursor rRNA from oocytes is capped by these enzymes, and we conclude that it has 5'-polyphosphate termini. Therefore, 40S RNA must represent the primary transcript of amplified X. laevis ribosomal DNA. The majority of 40S molecules with polyphosphate termini begin with the sequence (p)ppAAG. There is evidence, however, that the 5' terminus may be heterogeneous. The majority of all detectable initiation events were localized close to the region coding for the 5' end of the 40S RNA. No initiation sites were detected in the nontranscribed spacer, but an apparent initiation site in the middle of the transcribed region was also observed.

Animals↗
PubMed1976

Chromatin structure as probed by nucleases and proteases: evidence for the central role of histones H3 and H4.

We have examined the role by each histone in forming the structure of the nu-body. When DNAase I, DNAase II, trypsin and chymotrypsin attack chromatin, characteristic discrete DNA and protein digest fragments are produced. Using this restriction of accessibility as diagnostic for chromatin structure, we have examined complexes of DNA with virtually all possible combinations of histones. The results strongly support our previous conclusion (Camerini-Otero, Sollner-Webb, and Felsenfeld, 1976) that the arginine-rich histones are unique in their ability to create, with DNA a structure with many features of native chromatin. Acting together, slightly lysine-rich histones then modify this complex into one very similar to native chromatin. An analysis of the rate constants of staphylococcal nuclease digestion also confirms that the complex of H3, H4, and DNA is crucial to the structure of the nu-body.

Animals↗
PubMed1976

The organization of histones and DNA in chromatin: evidence for an arginine-rich histone kernel.

We have examined the role played by various histones in the organization of the DNA of the nucleosome, using staphylococcal nuclease as a probe of DNA conformation. When this enzyme attacks chromatin, a series of fragments evenly spaced at 10 base pair intervals is generated, reflecting the histone-DNA interactions within the nucleosome structure. To determine what contribution the various histones make to DNA organization, we have studied the staphylococcal nuclease digestion patterns of complexes of DNA with purified histones. Virtually all possible combinations of homogeneous histones were reconstituted onto DNA. Exhaustive digestion of a complex containing the four histones H2A, H2B,H3, and H4 yields a DNA fragment pattern very similar to that of whole chromatin. The only other combinations of histones capable of inducing chromatin-like DNA organization are H2A/H2B/H4 and those mixtures containing both H3 and H4. From an examination of the kinetics of digestion of H3/H4 reconstitutes, we conclude that although the other histones have a role in DNA organization within the nucleosome, the arginine-rich histone pair, H3/H4, can organize DNA segments the length of the nucleosome core in the absence of all other histones.

Animals↗
PubMed1975

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.

Animals↗
PubMed1974

Specific sites of interaction between histones and DNA in chromatin.

Staphylococcal nuclease digestion of purified chromatin from duck reticulocytes or calf thymus results in the production of a series of double-stranded DNA fragments of discrete molecular size, ranging from about 130 to 45 base pairs, which can be detected by polyacrylamide gel electrophoresis. Similar patterns of protected DNA fragments are obtained from limit digests of chromatin "reconstituted" from purified DNA and chromatin proteins. The results obtained with reconstituted material do not depend upon the origin of the DNA, which may be derived from a bacterial, viral, or homologous source. The specificity of the protective mechanism, therefore, resides in the structure of the bound histones, and probably not in any special nucleotide sequences present in the DNA. Removal of lysine-rich histones from chromatin before digestion results principally in disappearance from the digest of a DNA fragment about 130 base pairs long. Our preliminary results suggest that other elements of the digest pattern can be assigned uniquely to the remaining histone components. These results indicate that the binding of histones to DNA in chromatin involves a limited number of specific and very well defined contacts between protein and nucleic acid, which arise from structural properties of the histones.

Animals↗
PubMedDate not supplied

Expression of mouse and frog rRNA genes: transcription and processing.

This article summarizes a number of lines of investigation of rRNA gene expression that are ongoing in the laboratory. These studies focus on mouse and frog, two distant vertebrate species. One major conclusion is that the basic properties of rRNA gene expression appear remarkably well conserved in evolution, with only relatively minor perturbations between frog and mouse, contrary to the common interpretation of the species-selectively between mouse and human rDNA transcription (e.g., 1). This is true both for the process of rDNA transcription and for the subsequent rRNA processing event.

Animals↗