Generating tandem repeats by cloning with double initiator fragments.

The ability to generate tandem repeats of a DNA sequence has proven important for a large variety of studies of DNA structure and function. The most commonly used method to produce tandem repeats involves cloning of an oligomerized monomer sequence that contains asymmetric overlapping ends, but, in practice, this approach is inefficient because of the circularization of oligomers before they ligate into vector. Described here is a method that circumvents this problem by the use of two separate oligomerization reactions, each containing an initiator fragment onto which monomer polymerizes without circularization. Subsequent mixing of the two reactions permits circularization, generating a viable plasmid containing the sum of the added repeats from each reaction. A variation of this method is also demonstrated that permits the synthesis of constructs with a defined number of repeats.

TATA box occupancy in the SV40 transcription elongation complex.

In order to gain insight into requirements for template activation and commitment in mammalian transcription, TATA site occupancy was measured in native SV40 viral transcription complexes that were in the process of transcription elongation at the time of cell lysis. This was accomplished by quantifying resistance to restriction enzyme digestion of transcription complexes in nuclear lysate. The rate of cleavage at the TATA site of the late gene in the native complex was slower than that of a bare DNA control, both for wild-type virus and for a virus containing a TATA consensus sequence. These results suggest that the TATA site in the transcription elongation complex in vivo is occupied with transcription factor TBP/TFIID. When considered in light of previous work, these findings support a model in which transcription activation involves reinitiation from a promoter that contains both activator and TFIID bound in a stable complex.

Measurement of the linking number change in transcribing chromatin.

The in vivo-initiated, transcribing simian virus 40 (SV40) minichromosome was analyzed to determine its DNA linking number change, i.e. the difference between the linking number of the minichromosomal DNA and that of relaxed bare DNA. As part of this measurement, the linking number change due to the in vivo-initiated RNA polymerase II was determined, the first time a value for this quantity has been reported. The topological contribution of the polymerase was combined with values determined for constrained and non-constrained linking number contributions from the native transcription complex chromatin to yield the linking number change for the complex. The linking number change of the native non-transcribed SV40 minichromosome was independently determined and was found to be virtually the same as that for the chromatin of the transcription complex. This indicates that there is little difference between the two structures. The plausibility of several current models for the contribution of chromatin structure to transcription regulation is discussed in light of this finding.

Chromatin structure and factor site occupancies in an in vivo-assembled transcription elongation complex.

The chromatin structure specific to the SV40 late transcription elongation complex as well as the occupancy of several sites that bind transcription factors have been examined. These features have been determined by assessing blockage to restriction enzyme digestion. Cleavage specific to the elongation complex has been quantified using ternary complex analysis. This method involves radioactively labeling the complex by in vitro transcription followed by determining the extent of linearization by electrophoresis in an agarose gel. It was found that not only is the origin region devoid of nucleosomes, but there is also no stable factor occupancy at the BglI, SphI, KpnI and MspI restriction enzyme sites within this region. Thus these sites were cleaved to a high degree, meaning that the binding sites for a number of transcription factors, including OBP/TEF-1, TBP, DAP, as well as a proposed positioned nucleosome, are unoccupied in the native viral transcription elongation complex. The absence of these trans-acting factors from their respective binding sites in the elongation complex indicates that they bind only transiently, possibly cycling on and off during the transcription cycle. This finding implies that various forms of transcription complex are assembled and disassembled during transcription and thus supports a 'hit-and-run' model of factor function.

Topological measurement of protein-induced DNA bend angles.

A new method measures independently the changes in the DNA bend and winding angles that occur when a protein binds to its specific site in DNA. The procedure requires an investigation of the change in DNA topology induced when the protein binds to tandemly repeated sites of varying repeat length in circular DNA. This new method is superior to currently used methods because topology permits the measurements to be derived absolutely from first principles, and no comparison standards are required. The method is used to determine the bend and winding angles induced when catabolite gene activator protein binds to its site. The bend value obtained (69 +/- 4 degrees) is intermediate to those reported for two crystal forms of the complex.

Determination of occupancies of the SPH and GT-IIC transcription factor binding motifs in SV40: evidence for two forms of transcription elongation complex.

Occupancies of the SPH and GT-IIC sequence motifs in the native SV40 late transcription elongation complex were determined by assessing blockage to restriction enzyme cleavage. Cleavages specific to the transcription elongation complex were quantified by radioactive extension labeling and polymerase run-off analysis. The SPH motif was assayed by Sphl digestion and found to be unoccupied. In contrast, digestion with Pvull at the GT-IIC site was blocked in 36% of the complexes, indicating that approximately a third of the complexes are occupied by factor. This fractional occupancy indicates that there are at least two forms of SV40 late transcription elongation complexes, one form with the GT-IIC site occupied by a factor and another with the site vacant.

RNA polymerase locations in the simian virus 40 transcription complex.

Transcription complexes of simian virus 40 can be isolated from cells late in infection in a form that retains the ability to continue transcription in vitro. These complexes have been investigated previously to gain information about the nucleoprotein structure of a transcribing gene. However, several studies have reported that the RNA polymerase molecules in such complexes are located almost entirely at the 5' end of the transcription unit. This would indicate that these complexes cannot be those which were in the process of transcribing mRNA in the cell, because the transcribing complexes would be expected to contain polymerase that were distributed throughout the transcription unit at the time of extraction. Since this issue is important to the interpretation of studies which characterize the extracted complexes, we have determined the polymerase locations using two new approaches. The first employs a new application of a procedure in which the transcription complex DNA is radioactively tagged by transcription in vitro. The second is a new method, which analyzes run-off transcripts generated in vitro. Both methods demonstrate that the polymerases are distributed throughout the genome in the transcription complexes. This result indicates that these complexes were in the process of transcribing mRNA at the time of cell lysis.

Effects of histone acetylation on chromatin topology in vivo.

Recently a model for eukaryotic transcriptional activation has been proposed in which histone hyperacetylation causes release of nucleosomal supercoils, and this unconstrained tension in turn stimulates transcription (V. G. Norton, B. S. Imai, P. Yau, and E. M. Bradbury, Cell 57:449-457, 1989; V. G. Norton, K. W. Marvin, P. Yau, and E. M. Bradbury, J. Biol. Chem. 265:19848-19852, 1990). These studies analyzed the effect of histone hyperacetylation on the change in topological linking number which occurs during nucleosome assembly in vitro. We have tested this model by determining the effect of histone hyperacetylation on the linking number change which occurs during assembly in vivo. We find that butyrate treatment of cells infected with simian virus 40 results in hyperacetylation of the histones of the extracted viral minichromosome as expected. However, the change in constrained supercoils of the minichromosome DNA is minimal, a result which is inconsistent with the proposed model. These results indicate that the proposed mechanism of transcriptional activation is unlikely to take place in the cell.

Enhancer-activated plasmid transcription complexes contain constrained supercoiling.

It has been proposed that transcriptionally active chromatin contains totally unconstrained supercoiling. The results of recent studies have raised the possibility that this topological state is the property of highly transcribed genes. Since the transcription rate of RNA polymerase II genes can be dramatically increased by the presence of an enhancer, we have determined if the transcription complex of an enhancer-activated plasmid contains totally unconstrained supercoils. Following transfection into COS7 cells, the topology of the transcription complex DNA was determined directly by agarose gel electrophoresis. We find that an enhancer-activated plasmid transcription complex is supercoiled, and these supercoils remain following topoisomerase I treatment. Thus the transcribing complexes contain constrained supercoils, and the level of supercoiling suggests a nucleosome-like organization. However, we cannot rule out the possibility that unconstrained supercoils exist in addition to these constrained supercoils in the transcription complex in the cell.

T-antigen is not bound to the replication origin of the simian virus 40 late transcription complex.

Simian virus 40 tumor antigen (T-antigen) plays a central role in determining which gene is transcribed from viral DNA late in infection. Results from several studies have led to a model in which the binding of T-antigen to the viral origin of replication results in repression of transcription from the stronger early gene promoter and stimulation of transcription from the late gene promoter. We have tested this model by determining directly the occupancy of the T-antigen binding site in the origin of replication of the late transcription complex. Thus, viral transcription complexes were digested with BglI, a restriction enzyme that cuts in the viral replication origin. The enzyme cleaved 78(+/- 12)% of the late transcription complexes. Control experiments demonstrated that cleavage is blocked when T-antigen is bound to the origin site, that exogenously added T-antigen can bind to the site in the transcription complex, and that T-antigen is not released during isolation of the complex. These results indicate that most of the late transcription complexes do not have T-antigen bound to the origin site, and are therefore inconsistent with models that require this site to be occupied by T-antigen to maintain proper regulation of gene transcription late in infection.

Thermal unwinding of simian virus 40 transcription complex DNA.

Two long-standing questions in the control of eukaryotic gene expression have been how the structure of transcribing chromatin compares with that of nontranscribing chromatin and how chromatin structure differs among various eukaryotic organisms. We have addressed aspects of these two questions by characterizing the rotational flexibility of the DNA of the simian virus 40 (SV40) transcription complex. When transcription complex samples are incubated with topoisomerase at 0 degrees C or 37 degrees C, the DNA of the 37 degrees C sample is unwound by 1.8 turns relative to that of the 0 degrees C sample. This amount of unwinding is similar to that observed for bulk, untranscribed SV40 minichromosome DNA, indicating that the chromatin structure of a transcribed gene resembles that of a nontranscribed gene in the degree of constraint that it imposes on its DNA. However, this amount of unwinding differs substantially from the value observed for yeast plasmid chromatin DNA, suggesting that yeast chromatin differs significantly from mammalian chromatin in this fundamental property.

Immunoprecipitation of the simian virus 40 late transcription complex with antibody against T-antigen.

Tumor antigen (T-antigen) of simian virus 40 (SV40) has been shown to have a number of regulatory roles in both viral replication and early viral transcription. However, the nature of its role on late viral transcription remains unclear. We have analyzed for the presence of T-antigen on SV40 late viral transcription complexes which exhibit RNA polymerase II extension activity in vitro. Nuclear extract or glycerol gradient-isolated transcription complexes were treated with either polyclonal or monoclonal antibodies, and the amount of extension activity that could be immunoprecipitated was determined. Anti-T antibody derived from hamster ascites as well as the anti-T monoclonal antibodies PAb 102, 109, 416, and 419 all precipitated 12-29% of viral transcription complex activity. Immunoprecipitation resulted in significant enrichment of transcription complex activity relative to bulk minichromosomes, indicating a preferential association of T-antigen with the late viral transcription complex. This is the first direct demonstration of the presence of T-antigen on the SV40 late transcription complex. Furthermore, the immunoprecipitated transcription complexes exhibited a salt dependence of their in vitro extension activity which was distinct from that of the total complex population, indicating that T-antigen is present on a specific subclass of transcription complexes.

Topological characterization of the simian virus 40 transcription complex.

We have used sedimentation analysis as well as agarose gel electrophoresis to characterize the topological state of the DNA of the Simian Virus 40 (SV40) transcription complex. We found that the complex DNA contained constrained topological tension, presumably resulting from nucleosome-like structures, but no detectable unconstrained (i.e., relaxable) topological tension. These results contradict previous conclusions that the SV40 transcription complex contains only unconstrained topological tension. Our findings are also the opposite of what has been proposed to be the case for the 5S gene analyzed in Xenopus oocytes. Thus the proposal that expression from the 5S gene is associated with substantial topological tension is not valid for expression from the SV40 late gene.

Optimization of polyacrylamide gel electrophoresis conditions used for sequencing mixed oligodeoxyribonucleotides.

We investigated two components of the polyacrylamide gel electrophoresis system used for sequencing DNA to improve the system for sequencing synthesized oligodeoxyribonucleotides containing positions of degeneracy. First, we varied the ratio of methylene-bis-acrylamide (MBA) to acrylamide from that commonly used in DNA sequencing gels (1% MBA:19% acrylamide). A moderate increase in the MBA:acrylamide ratio proves optimal when sequencing is used to confirm that a synthesized fragment contains equivalent stoichiometries of the different nucleotides at a given position of degeneracy. Such information is particularly important if the degenerate oligodeoxyribonucleotide preparation is to be employed as a hybridization probe. A further increase in the MBA:acrylamide ratio (3% MBA:19% acrylamide) produces a gel in which the sequence of an oligodeoxyribonucleotide mixture containing several positions of degeneracy can be read most easily. Increasing the MBA acrylamide ratio suppresses the effect of base composition on electrophoretic mobility of a fragment. Second, we investigated the use of a Tris-citrate buffer system in place of the standard Tris-borate system. We found the Tris-citrate system to be significantly more effective in preventing discontinuities in the banding pattern of the smaller fragments. Finally, we show that high MBA gels are also effective in resolving mixtures of oligoribonucleotides such as those produced by T1 ribonuclease digestion of small RNAs.

Affinity isolation of RNA polymerase II on amanitin-Sepharose.

We report here the first case of an affinity isolation of eukaryotic RNA polymerase II. The procedure employs an affinity matrix composed of alpha-amanitin coupled to Sepharose 4B via a ten atom spacer. RNA polymerase II from either calf thymus or wheat germ binds to the amanitin-Sepharose, as indicated by subsequent elution with sodium dodecylsulfate-containing buffer and analysis by polyacrylamide gel electrophoresis. The specificity of binding is demonstrated by the fact that when the enzyme is preincubated with 1 microgram/ml of free alpha-amanitin, subsequent binding to the amanitin-Sepharose is abolished. Elution methods that should permit the recovery of active enzyme from the column are discussed.

Photoreactivation of amanitin-inhibited RNA polymerase II.

The fungal toxin alpha-amanitin binds very tightly to RNA polymerase II (Kd approximately 10(-9) M) and inhibits the polymerizing activity of the enzyme. However, it has been unclear from previous studies whether or not this inhibition can be reversed. We show in this communication that it is possible to reverse the amanitin inhibition of polymerase by using a novel photoreactivation technique. When the inactive amanitin-wheat germ polymerase II complex is exposed to ultraviolet irradiation of a wavelength which is absorbed by the amanitin but not by the polymerase (monochromatic 314-nm irradiation), the enzyme recovers virtually all of the activity that an uninhibited control exhibits. Ultraviolet irradiation of a wavelength which is not absorbed significantly by the amanitin (monochromatic 350-nm irradiation) does not reactivate the inhibited polymerase. This ability to photoreactivate polymerase is discussed with respect to the mechanism of action of amanitin inhibition. Also discussed is the use of this technique for photoelution of an amanitin-Sepharose affinity column which we are developing for the isolation of transcriptionally active chromatin.

The helical periodicity of DNA on the nucleosome.

The precise number of base pairs per turn of the DNA double helix in the nucleosome core particle has been the subject of controversy. In this paper the positions of nuclease cutting sites are analysed in three dimensions. Using this midpoint of the DNA on the nucleosome dyad as origin, the cutting site locations measured along a strand of DNA are mapped onto models of the nucleosome core containing DNA of different helical periodicities. It is found that a helical periodicity of 10.5 base pairs per turn leads to cutting site positions which are sterically inaccessible. In contrast, a periodicity of 10.0 base pairs per turn leads to cutting site positions which are not only sterically sound, but which fall into a pattern such as would be expected when the access of the nuclease to the DNA is restricted by the presence of the histone core on one side and of the adjacent superhelical turn of DNA on the other. As proposed earlier by us (1), a value for the helical periodicity close to 10 base pairs per turn on the nucleosome, taken together with a periodicity close to 10.5 for DNA in solution - a value now established - resolves the so-called linkage number paradox.

DNase II digestion of the nucleosome core: precise locations and relative exposures of sites.

The precise locations and relative exposures of the DNase II-accessible sites in the nucleosome core DNA are determined using techniques previously employed for the enzyme DNase I. It is found that there are a number of similarities between the site exposure patterns for the two enzymes but that in general the DNase II seems to discriminate less among adjacent sites' accessibilities than does DNase I. The two enzymes attack essentially the same positions in the DNA, the average difference between the precise location of the site being less than one-half base for the two enzymes. Such close similarities in the digestion patterns of two enzymes with such different mechanisms of scission show that the patterns reflect the structure of the nucleosome core and not merely the properties of the particular enzyme used.