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E B Kmiec

Publications and source records attributed to E B Kmiec.

At least 73 records · Page 4Linked to original sources
PubMed1990

cis-acting enhancement of RNA polymerase III gene expression in vitro.

The Xenopus laevis S-150 cell-free extract catalyzes in vitro transcription of several RNA polymerase III genes. Among these are the Xenopus 5S RNA gene (somatic type) and the Xenopus methionine tRNA gene. In this report we present an analysis of the transcriptional activity of these two genes either in trans-competition experiments or when the genes are co-localized in the same circular plasmid. In the "cis" arrangement, elevated levels of 5S and tRNA gene expression are observed, which are dependent on the relative orientation of the two genes (convergent or in tandem) and the distance between them. The results of these analyses reveal important parameters affecting the expression of juxtaposed genes.

Animals↗
PubMed1990

Purification and characterization of a type-I topoisomerase from cultured tobacco cells.

Cultured tobacco (Nicotiana tabacum, var Xanthi) cells contain a topoisomerase that removes positive and negative supercoils from DNA. The enzyme has an estimated molecular mass of 30,000 daltons under denaturing conditions, but may exist as a multimeric protein in the native state. Activity is enhanced significantly by either MgCl(2) or CaCl(2), but other divalent cations are much less effective in stimulating DNA relaxation. The purified enzyme acts by altering the linking number in topological steps of one and is inhibited by berenil or camptothecin, not novobiocin. Taken together, these data identify this enzyme as a type I topoisomerase.

Journal Article↗
PubMed1990

A glucocorticoid response element enhances transcription of a methionine tRNA gene in cis and trans.

In vitro transcription experiments using a Xenopus laevis cell-free extract have demonstrated that a DNA fragment containing a glucocorticoid response element (GRE) significantly enhances the expression of a methionine tRNA gene. This stimulation can be observed when the element is located in cis or trans. In the cis configuration, the element can be located 2900 basepairs from the gene and still display transcriptional enhancement. In trans, the enhancement can occur at a low element to template molecular ratio. The nucleosome positioning and chromatin structure near the tRNA gene appear to be unaffected by the presence of the long terminal repeat of the mouse mammary tumor virus which contains the GRE. Taken together, these data suggest that GREs can stimulate transcription of hormonally unresponsive genes, perhaps by providing a "molecular sink" to which inhibitors may bind. Further experimentation may reveal a correlation between these in vitro studies and in vivo gene regulation.

Animals↗
PubMed1990

Assembly of transcriptionally active chromatin in vitro: a possible role for topoisomerase II.

Some models of in vitro chromatin assembly suggest a biphasic molecular mechanism. The first phase, nucleosome formation, is comprised of the formation of histone-DNA complexes which mature into a canonical nucleosome structure. The second phase represents the process by which these nucleosomes become properly spaced with a regular periodicity on the DNA. In this report, we examine the role of DNA topoisomerases in the latter phase of chromatin assembly. To study this process, we use a Xenopus laevis cell-free extract, which assembles quantitative amounts of chromatin on circular DNA templates, and the type II topoisomerase-specific antitumor drugs VM-26 and endrofloxicin. Our results suggest that nucleosome formation is unaffected by the presence of VM-26 or endrofloxicin. However, periodic spacing of nucleosomes is inhibited significantly by these drugs. In the absence of proper chromatin assembly, circular DNA molecules are processed into nucleoprotein complexes which are transcribed poorly. Taken together, these results indicate that the antitumor drugs VM-26 and endrofloxicin influence gene expression indirectly by blocking the periodic spacing of nucleosomes.

Adenosine Triphosphate↗
PubMed1989

Changes in DNA topology can modulate in vitro transcription of certain RNA polymerase III genes.

The role of DNA supercoiling in eukaryotic gene expression is not fully understood. The objective of this study was to examine the regulation of in vitro chromatin assembly by topological alterations in the DNA template using a cell-free extract from Xenopus laevis oocytes (S-150). The results suggest that input DNA topology may be a determining factor in controlling the transcriptional activity of the Xenopus tRNA and one particular 5S gene. When the input topology is supercoiled, high levels of transcription are observed, whereas input relaxed DNA is transcribed to a much lower extent. Transcription from an input relaxed template is stimulated by the addition of supercoiled nonspecific, vector DNA. Furthermore, in direct competition experiments, supercoiled DNA molecules were shown to be transcriptionally dominant over relaxed DNA molecules. Taken together, these data suggest that the efficiency with which a repressor or activator binding protein interacts with DNA may be significantly influenced by the topological status of its target. We demonstrate that modulation of reaction parameters which alter the normal topological processing events catalyzed by the S-150 can dramatically influence the level of gene expression.

Animals↗
PubMed1989

DNA superhelicity enhances the assembly of transcriptionally active chromatin in vitro.

Using an in vitro chromatin assembly system, we analyzed the influence of DNA superhelicity on the development of transcriptionally active minichromosomes. Plasmid DNA molecules containing either a Xenopus borealis 5S RNA gene or an X. laevis methionine tRNA gene were utilized as templates for the assembly of chromatin. Both plasmids were processed into active minichromosomes if introduced as supercoiled molecules into the extract (S-150). The degree of superhelicity is a determining factor in the assembly of active chromatin. Molecules containing varying superhelical densities were processed into minichromosomes with different transcriptional activities. The absence of supercoils leads to the assembly of chromatin with substantially lower transcriptional activity. Assembled minichromosomes are stable enough to be isolated by sucrose gradient centrifugation while retaining their transcriptional phenotype. The formation of nucleosomes with a periodic spacing occurred with the same efficiency and to the same degree regardless of the initial DNA topology. Hence, a determining factor in the development of transcriptionally active chromatin may be the initial superhelicity of the DNA molecule to which activator (trans-acting factors) or repressor (histones) proteins bind. Once the chromatin assembly process has begun, the transcriptional activity of the resulting minichromosome may already have been determined.

Animals↗
PubMed1989

Assembly of transcriptionally inactive chromatin in vitro.

We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.

Animals↗
PubMed1989

Studies on the ATP requirements of in vitro chromatin assembly.

To gain a more complete understanding of the process of in vitro chromatin assembly, an examination of the energy requirements of nucleosome formation must be undertaken. The experiments outlined in this manuscript address this issue by making use of the Xenopus laevis S-150 cell-free extract. The S-150 catalyzes chromatin assembly on circular DNA templates dependent either on the exogenous addition of ATP or regeneration of endogenous ATP. We define two distinct, but temporally ordered, phases of the overall process. The first, nucleosome formation, occurs in the presence of endogenous levels of ATP, while the second phase, chromatin assembly, which we define as the development of properly spaced nucleosomes, requires a higher level of ATP. Both phases lead to a distribution of molecules with similar superhelical densities. Taken together, these data suggest that chromatin assembly may consist of two distinct steps differing in their strategy cofactor requirement. The experiments presented in this manuscript support the concept that nucleosomes first assemble, perhaps randomly, on the DNA and are gradually matured into a canonical chromatin structure with periodic spacing.

Adenosine Triphosphate↗
PubMed1988

Studies on DNA topoisomerase activity during in vitro chromatin assembly.

The in vitro assembly of chromatin, promoted by the Xenopus cell-free extract (S-150), can be inhibited by oxolinic acid and to a lesser extent by nalidixic acid. Both of these antibiotics have been shown to block the activity of the specialized type II Topoisomerase, bacterial DNA Gyrase. Oxolinic acid induces a DNA cleavage by Micrococcal Nuclease at specific sequences in the multiple cloning vector pGEM-4. Nalidixic acid does not inhibit DNA supercoiling, but does diminish the extent of chromatin formation achieved by the S-150 on circular DNA templates. The Topoisomerase I inhibitor, berenil, does not inhibit extensive chromatin assembly, although it does diminish the level of supercoiling. Taken together, these results suggest that both topoisomerases play a role in the assembly process. Topoisomerase I may catalyze both the introduction of unconstrained supercoils into relaxed DNA and the formation of monosomes, while Topoisomerase II may promote extended chromatin assembly.

Animals↗
PubMed1986

The role of DNA-mediated transfer of TFIIIA in the concerted gyration and differential activation of the Xenopus 5S RNA genes.

The transcription factor of the 5S RNA gene, TFIIIA, induces gyration of oocyte- and somatic-type 5S DNA plasmids in Xenopus oocyte extracts, but oocyte 5S gyration requires a 5-fold higher TFIIIA concentration than somatic 5S gyration. Concomitant with the differential gyration at intermediate TFIIIA concentrations, the oocyte genes are repressed and the somatic genes become activated, a situation that mimics the one seen in Xenopus somatic cells. Data obtained with plasmids immobilized in agarose indicate that TFIIIA finds its site via a DNA-mediated transfer mechanism, and that all-or-none gyration is a consequence of TFIIIA transfer between 5S DNA sites. Based on these results, we present a model that explains the differential all-or-none activation of the 5S RNA genes.

Animals↗
PubMed1986

Homologous pairing of DNA molecules by Ustilago rec1 protein is promoted by sequences of Z-DNA.

Plasmids containing Z-DNA stretches can be paired and linked by combined action of Ustilago rec1 protein and topoisomerase. The product formed is a hemicatenated dimer in which two DNA rings are topologically intertwined at a region of homology. Superhelicity governs the reaction. Formation of linked product is coupled with formation of Z-DNA in the plasmid, a process dependent on the superhelix density. Pairing appears to initiate within the Z-DNA sequence, not at the unwound B-Z junction. The reaction can be blocked by a Z-DNA-specific binding protein, namely Z-DNA antibody. Plasmids with alternating Z-DNA dG-dC sequences at different sites on otherwise homologous molecules can be linked at the dG-dC sequences. However, a plasmid with a (dG-dC)n.(dG-dC)n Z-DNA stretch cannot be linked with a plasmid containing a (dG-dT)n.(dC-dA)n Z-DNA stretch.

Antigen-Antibody Complex↗
PubMed1986

Gyration is required for 5S RNA transcription from a chromatin template.

We have assembled transcriptionally active chromatin on 5S DNA plasmids by using a Xenopus oocyte supernatant and the 5S-specific transcription factor IIIA (TFIIIA). In this system, the 5S RNA gene is accurately transcribed at a rapid rate of 50 transcripts per gene per hr. By following the time course of RNA synthesis during chromatin assembly, the dose response to TFIIIA addition, and the effect of novobiocin on the assembled nucleoprotein, we show that there is a strict correlation between transcriptional activity and the generation of torsionally strained DNA supercoils in "dynamic chromatin." Transcription cannot be the cause of the dynamic structure, because the assembly of this chromatin is unaffected by alpha-amanitin levels that completely block RNA polymerase III. Surprisingly, the dynamic chromatin remains transcriptionally active after relaxation with DNA topoisomerase I, which implies that the essential parameter for chromatin transcription is gyration per se, and not its effect on DNA topology.

Animals↗
PubMed1986

The positive transcription factor of the 5S RNA gene proteolyses during direct exchange between 5S DNA sites.

We have examined the association, dissociation, and exchange of the 5S specific transcription factor (TFIIIA) with somatic- and oocyte-type 5S DNA. The factor associates faster with somatic than with oocyte 5S DNA, and the rate of complex formation is accelerated by vector DNA. Once formed, the TFIIIA-5S DNA complex is stable for greater than 4 h in the absence of free 5S DNA, and its dissociation is identical for somatic and for oocyte 5S DNA. In the presence of free 5S DNA, the factor transfers promptly from the complex to the free 5S DNA site. Unexpectedly, the direct exchange of factor between 5S DNA sites leads to proteolysis at the C-terminal arm of TFIIIA.

Animals↗
PubMed1985

Left-handed DNA and the synaptic pairing reaction promoted by Ustilago rec1 protein.

Left-handed Z-DNA binds tightly to Ustilago rec1 protein. The binding reaction is strongly dependent on ATP, but complexes formed are rapidly dissociated by ADP. The parallel between the kinetics of Z-DNA binding and the synaptic pairing reaction leading to paranemic joint molecules suggests that formation of nascent heteroduplex structures in recombination is coupled with formation of left-handed Z-like DNA on the protein. Equilibrium and kinetic studies show that rec1 protein appears to have a strong Z-DNA binding site that binds Z-DNA 75 times tighter than the B form of the DNA. We propose that DNA with a structure approximated best by a left-handed Z-DNA conformation is a key intermediate in homologous pairing promoted by rec1 protein.

Animals↗
PubMed1985

The positive transcription factor of the 5S RNA gene induces a 5S DNA-specific gyration in Xenopus oocyte extracts.

We report that TFIIIA, the positive transcription factor of the 5S RNA gene, induces DNA gyration in Xenopus oocyte extracts. The reaction uses one molecule of TFIIIA per molecule of DNA and is highly specific for 5S DNA plasmids. DNA gyration also requires the oocyte supernatant, ATP, and Mg2+, and is inhibited by novobiocin, suggesting that it is catalyzed by a type II DNA topoisomerase. The chromatin assembled with TFIIIA is dynamic and rapidly relaxed by novobiocin; the chromatin assembled without TFIIIA is static and unaffected by novobiocin. The torsionally strained DNA is produced in a novel concerted reaction: all of the 5S DNA molecules gyrate at TFIIIA-5S DNA ratios equal to or above 1, and none of them gyrate at TFIIIA-5S DNA ratios below 1. We discuss the biological implications of this eukaryotic DNA gyration.

Adenosine Triphosphate↗
PubMed1984

Hysteretic regulation of Ustilago rec1 protein during synapsis.

rec1 protein slowly loses activity in synaptic pairing reactions. The explanation for this result would appear to be that the overall reaction is limited by a slow transition of the protein to an inactive state. When preincubated with single-stranded DNA and ADP, rec1 protein forms a dead-end complex that is unable to promote homologous pairing. Its pairing activity can be restored by lowering the temperature for several minutes or can be maintained if duplex DNA sharing no homology with the single-stranded DNA is also included. These results suggest a mechanism for attenuating pairing activity during recombination. After recognition of homologous sequences, rec1 protein undergoes a slow, ligand-induced conformational change to an inactive form.

Adenosine Diphosphate↗
PubMed1984

Synapsis promoted by Ustilago rec1 protein.

Ustilago rec 1 protein pairs homologous DNA molecules by promoting both synapsis and strand transfer. Complexes formed with rec 1 protein and a homologous combination of single-stranded and duplex DNA that appear to be synaptic structures can be detected by use of a nitrocellulose filter assay. The nascent heteroduplex formed during synapsis is a paranemic joint in which the single-stranded DNA pairs, but does not interwind, with its complement in the duplex molecule. Formation of the paranemic joint is accompanied by duplex unwinding and genesis of left-handed Z-DNA.

Adenosine Diphosphate↗