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M Dunaway

Publications and source records attributed to M Dunaway.

18 recordsLinked to original sources
PubMed1999

Modeling transcriptional regulation using microinjection into Xenopus oocytes.

Transcriptional regulation is a complex process that requires cooperation between specific DNA sequence elements, the DNA-binding proteins that bind to these sequences, the general transcriptional machinery, and chromatin. Oocyte microinjection offers a technique to study the integrated transcription process while still providing the opportunity to experimentally perturb this process. We describe here techniques for manipulating DNA templates and the protein complement of the oocyte to study multiple facets of transcriptional regulation. We present sample results showing that the GAL4-VP16 fusion activator is sensitive to distance in constructs containing only a minimal promoter, but can activate transcription at greater distances when proximal promoter elements are present.

Animals↗
PubMed1998

The scs and scs' insulator elements impart a cis requirement on enhancer-promoter interactions.

The Xenopus rRNA enhancer activates its cognate promoter when the two elements are placed on opposite rings of dimeric catenanes. Here we show that when scs elements flank either the enhancer or promoter in catenanes, the enhancer cannot activate the promoter on the ring in trans. A series of catenanes containing different permutations of the insulators, enhancer, and promoters shows that when insulators are present, the enhancer is permitted to a activate the promoter only when both elements are on the same piece of DNA with no intervening insulator. These results suggest that insulators have the potential to block enhancer-promoter interactions between chromosomes and between independent topological domains within a chromosome.

Animals↗
PubMed1997

The activity of the scs and scs' insulator elements is not dependent on chromosomal context.

Sequence elements that protect a reporter gene from chromosomal position effects or that block enhancer-activated transcription are called insulators. Using a plasmid-based microinjection assay with Xenopus laevis oocytes, we show that the heterologous Drosophila melanogaster scs and scs' insulator elements do not require chromosomal context to block enhancer-activated transcription. A single insulator element partially blocks enhancer-activated transcription, indicating that each element operates independently rather than as part of a pair. Deletion analysis of the 1.8-kb scs element identified a 220-bp fragment from one of the DNase I-hypersensitive regions that has full blocking activity in the oocyte assay. This fragment corresponds to the critical region of the scs mapped in previous studies with Drosophila. A time course of transcription shows that the scs blocks enhancer-activated transcription as early as transcription can be detected, about 30 min after injection. Complete assembly of the DNA template into nucleosomes requires 4 h. The scs and scs' sequences do not block site-specific recombination by FLP recombinase, implying that insulators do not operate by a general mechanism that physically sequesters the DNA. These data are most consistent with a model for insulator action in which direct interaction between the insulator and either the enhancer or promoter confers directionality to enhancer-activated transcription.

Animals↗
PubMed1997

The repeat organizer, a specialized insulator element within the intergenic spacer of the Xenopus rRNA genes.

We have identified a novel activity for the region of the intergenic spacer of the Xenopus laevis rRNA genes that contains the 35- and 100-bp repeats. We devised a new assay for this region by constructing DNA plasmids containing a tandem repeat of rRNA reporter genes that were separated by the 35- and 100-bp repeat region and a rRNA gene enhancer. When the 35- and 100-bp repeat region is present in its normal position and orientation at the 3' end of the rRNA reporter genes, the enhancer activates the adjacent downstream promoter but not the upstream rRNA promoter on the same plasmid. Because this element can restrict the range of an enhancer's activity in the context of tandem genes, we have named it the repeat organizer (RO). The ability to restrict enhancer action is a feature of insulator elements, but unlike previously described insulator elements the RO does not block enhancer action in a simple enhancer-blocking assay. Instead, the activity of the RO requires that it be in its normal position and orientation with respect to the other sequence elements of the rRNA genes. The enhancer-binding transcription factor xUBF also binds to the repetitive sequences of the RO in vitro, but these sequences do not activate transcription in vivo. We propose that the RO is a specialized insulator element that organizes the tandem array of rRNA genes into single-gene expression units by promoting activation of a promoter by its proximal enhancers.

Animals↗
PubMed1996

DNA length is a critical parameter for eukaryotic transcription in vivo.

The organization of eukaryotic chromosomes into topological domains has led to the assumption that DNA topology and perhaps supercoiling are involved in eukaryotic nuclear processes. Xenopus oocytes provide a model system for studying the role of DNA topology in transcription. Linear plasmid templates for RNA polymerases (Pols) I and II are not transcribed in Xenopus oocytes, while circular templates are transcriptionally active. Here we show that circularity is not required for transcription of Pol I or Pol II promoters if the linear template is sufficiently long (> 17 to 19 kb). The Xenopus rRNA (Pol I) promoter is active in central positions on a long linear template but is not transcribed when located near an end. Because supercoils generated by transcription could be retained by viscous drag against the long template, these results are consistent with a supercoiling requirement for this promoter. Surprisingly, the herpes simplex virus thymidine kinase (Pol II) promoter is active even 100 bp from the end of the long template, indicating that template length fulfills a critical parameter for transcription that is not consistent with a supercoiling requirement. These results show that DNA length has unrecognized importance for transcription in vivo.

Animals↗
PubMed1993

Local domains of supercoiling activate a eukaryotic promoter in vivo.

Experiments correlating template topology with transcriptional activity suggest that DNA topology plays a role in eukaryotic gene expression. Linear templates transfected into cultured cells produce far fewer transcripts than do circular transcription templates, and no transcripts can be detected from linear templates injected into Xenopus oocytes. Further, when transcriptionally active circular templates in Xenopus oocytes are linearized by injection of a restriction enzyme, transcription dramatically decreases. Here we show that transcription by phage T7 RNA polymerase from a divergent promoter can partially replace the requirement for circular Xenopus ribosomal RNA transcription templates in Xenopus oocytes. Supercoiled domains can apparently be generated on short pieces of DNA having no known sequences that result in association with the nuclear architecture, suggesting that localized, transient domains of supercoiling fulfil the minimum topological needs for Xenopus rRNA transcription in vivo.

Animals↗
PubMed1990

Reprogramming of the transcriptional machinery in Xenopus oocytes by injection of mouse poly(A)+ RNA.

We have devised an assay for species-specific transcription factors for the mouse rRNA gene by exploiting the ability of Xenopus oocytes to transcribe injected DNA and translate mRNAs. When mouse rRNA genes are microinjected into Xenopus oocytes, they are not transcribed. We show here that transcription of mouse rRNA genes is supported when mouse mRNAs are injected before the transcription template is injected, indicating that the necessary transcription factors are translated in the oocyte and are available to transcribe an appropriate template. The use of this assay in cloning genes for transcription factors is discussed.

Animals↗
PubMed1990

Inhibition of topoisomerase II does not inhibit transcription of RNA polymerase I and II genes.

Injection of VM-26 (teniposide) into Xenopus oocytes inhibits the activity of topoisomerase II but does not inhibit transcription by RNA polymerases I and II. A specific assay for topoisomerase II, resolution of catenated DNA molecules into product rings, was used to quantitate VM-26 inhibition in vivo. When catenanes were injected without VM-26, about 60% of them were separated into product rings in the first 5 min after injection, and decatenation of the remainder was complete within 15 min. When VM-26 was coinjected, 60% of the catenanes were separated into product rings in the first 5 min after injection, but the remaining 40% were stable over the next 40 min. At 1 h after injection catenanes were no longer detected in the gel analysis, but the increasing numbers of linear product rings indicated that topoisomerase II continued to be inhibited by VM-26. These results suggest that a short lag of approximately 5 min is required for VM-26 to inhibit topoisomerase II and that after this initial period topoisomerase II is inhibited by more than 90%. There was no detectable decrease in transcription of injected rRNA and thymidine kinase (TK) genes or in the activity of the rRNA enhancer when these transcription templates were coinjected with VM-26. The time required for assembly of injected DNA into chromatin doubled in the presence of VM-26.

Animals↗
PubMed1989

Transactivation of the Xenopus rRNA gene promoter by its enhancer.

A key question concerning the mechanism of transcriptional activation by enhancers is about the role of the DNA that connects the enhancer to the promoter. The linking DNA will be important if a regulatory protein(s) binds to the enhancer and then tracks or slides along the DNA to the promoter, or if, on binding, the protein(s) alters the topological state of the DNA. By contrast, if the linking DNA loops out to allow the formation of a promoter-enhancer complex, or if the enhancer increases the local concentration of a transcription factor, co-linearity of the promoter and the enhancer will not be strictly required. In Xenopus laevis, the transcription of the ribosomal RNA genes is stimulated by an enhancer composed of repetitive sequences in the intergenic spacer regions. These repetitive elements contain 60 or 81 base pairs, and their activity is relatively independent of their position and orientation. When the enhancer and promoter sequences are each located on separate DNA molecules, however, the enhancer is no longer able to augment transcription. We have now tested whether or not this apparent requirement for having the enhancer and promoter in cis can be overcome by keeping them in close proximity while locating them separately on different molecules. This was achieved by generating multiply intertwined, dimeric-catenanes in which the enhancer and promoter were located in trans on different rings. By injecting these catenanes into frog oocytes and measuring the activity of the enhancers in a series of competition assays, we were able to demonstrate that such enhancers can augment transcription in vivo.

Animals↗
PubMed1989

A transcription factor, TFIS, interacts with both the promoter and enhancer of the Xenopus rRNA genes.

An activity that binds sequence specifically to the enhancer of the Xenopus laevis rRNA genes has been highly purified. This activity stimulates transcription of coinjected rRNA templates in Xenopus oocytes and has been named TFIS, as it binds to the enhancer sequences within the intergenic spacer. In addition to its enhancer binding activity, TFIS binds to the promoter of the Xenopus rRNA genes, as predicted by models for enhancer action. DNase I footprinting on promoter mutants suggests that there are three TFIS-binding sites between -70 and -240 and that TFIS binding is unusually tolerant of mutations. The large region of protein-DNA interaction and the occurrence of DNase I enhancements at integral multiples of the helical repeat are consistent with the promoter and enhancer DNA wrapping around TFIS.

Animals↗
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.

Animals↗
PubMed1983

Spacer regulation of Xenopus ribosomal gene transcription: competition in oocytes.

Xenopus laevis ribosomal gene plasmids bearing different length spacers were injected into oocyte nuclei in competition with each other. The spacer has two basic effects on transcription from the gene promoter. First, if the competing pair have unequal spacer lengths, the gene promoter attached to the longer spacer is always dominant in transcription (the competition effect). Second, as the total amount of spacer in the reaction increases, the total amount of transcription decreases (the sink effect). Both the competition and the sink effect are attributed to sequence elements that are 60 or 81 bp long, which are present in multiple copies in the spacer and are related in sequence to part of the gene promoter. The 60/81 bp elements confer competitive dominance in either orientation. A model which explains both the competition and the sink effects is discussed in which the 60/81 bp elements are attraction sites for a factor(s) which is needed to activate the gene promoter.

Animals↗
PubMed1980

DNA binding characteristics of lactose repressor and the trypsin-resistant core repressor.

The nonspecific DNA binding capacity of repressor protein has been assessed by boundary sedimentation of repressor and calf thymus DNA fragmented by shearing and by nitrocellulose ultrafiltration employing labeled lambdaplac DNA in the presence of inducer concentrations sufficient to insure dissociation of repressor from the operator region of the DNA. These methods gave values in good agreement with values previously reported in the literature. The association constants for the interaction of repressor with operator DNA fragments and lambdaplac DNA have been measured and found to differ by approximately 100-fold at low salt concentrations, but the difference decreases to 4-fold at salt concentrations near the physiological value. The equilibrium association constant for the repressor-operator DNA fragment is significantly less sensitive to salt concentration than the corresponding constant for lambdaplac DNA. Inducer decreases the salt concentration dependence of repressor-operator DNA fragment only slightly. Measurement of the association constants for the interaction of the trypsin-resistant core protein with operator DNA fragment and lambdaplac DNA indicate that the core protein binds to the two DNA's with the same affinity. This result contrasts with the differential affinity of intact repressor for these two DNA's. In addition, the core protein association constants for operator DNA fragment and lambdaplac DNA display minimal dependence on the salt concentration. These results suggest a role for nonionic interactions in the binding of core protein to operator DNA.

Animals↗
PubMed1980

Kinetic studies of inducer binding to lac repressor.operator complex.

The rates of binding of inducer molecules to the lactose repressor protein are significantly affected by the presence of bound operator DNA fragments. The association rate is decreased from 4.6 x 10(4) M-1S-1 to 1.0 x 10(4) M-1S-1 by the presence of saturating amounts of operator DNA fragments. The inducer dissociation rate was measured by dilution of repressor . operator . inducer complexes and by displacement of the sugar molecule from repressor by the binding of operator DNA. The value for the dissociation rate in the presence of bound DNA was 0.8 S-1; this is 4-fold greater than the inducer dissociation rate measured in the absence of operator. These kinetic results suggest that repressor should exhibit a 20-fold lower affinity for inducer when operator DNA fragments are bound; this prediction is in agreement with previous equilibrium measurements. Measurement of the rate of operator fragment dissociation from repressor using a nitrocellulose filter assay yields a value of 0.04 S-1 in the absence of inducer; this dissociation rate is too rapid to measure in the presence of bound inducer molecules. Calculations assuming that the DNA-protein association rate is unaffected by sugar binding suggest that the rate of dissociation of DNA from repressor containing 4 bound inducer molecules may be as high as 40 S-1. Determination of the apparent association rate for inducer binding to operator DNA fragments indicates that DNA molecules do dissociate from the protein . operator . inducer complex at intermediate stages during the sugar binding reaction. The exact point at which dissociation occurs at a complex function of the concentrations of operator and inducer and the relationship between the rates for sugar binding and for operator fragment dissociation which are comparable for some of the protein species.

Allosteric Regulation↗
PubMed1980

Hybrid tetramers of native and core lactose repressor protein. Assessment of operator and nonspecific DNA binding parameters and their relationship.

Hybrid tetramers of lac repressor and its trypsin-resistant core protein were produced by mild proteolytic digestion and isolated by chromatography on phosphocellulose. These tetramers were used in binding studies to probe the relationship of the NH2 terminus and core domains in operator DNA binding and to explore subunit participation in both operator and nonspecific DNA binding. The purity of each tetramer was demonstrated by several lines of evidence, including physical characterization and measurement of the binding activities of the hybrid tetramer preparations. Each tetramer displayed measurable operator binding activity, and the dissociation constants of the tetramers for lambdaplac DNA and a 29 base operator fragment increase with the loss of each NH2 terminus. These studies strongly suggest that each NH2 terminus of the repressor interacts with the operator DNA, although the four NH2 termini do not appear to make equal contacts. The contributions of the NH2 terminus to operator binding do no appear to be identical with those for nonspecific DNA binding. In addition, the binding of the NH2 termini to nonspecific DNA can be described by postulating four identical, noninteracting sites. Although the rate of dissociation of the repressor-operator is increased by a factor of 2 upon removal of each NH2 terminus, calculations using the measured dissociation rate and equilibrium dissociation constant indicate that the rate of association of repressor to operator is decreased approximately 5-fold. These data suggest that the contacts made by the NH2 termini may greatly facilitate association with DNA. Competition studies of nonspecific DNA with operator DNA binding confirm the existence of two operator DNA binding sites on each tetramer and suggest that the contacts of a single NH2 terminus are not identical for the two operator binding sites.

Binding, Competitive↗
PubMed1980

Model for lactose repressor protein and its interaction with ligands.

A model is presented for the structure of the lactose repressor protein and for its interaction with inducer, operator DNA, and nonspecific DNA. The proposed structure is based on experimental evidence from this laboratory and from the literature and is offered as an integration of the available data on this system. Features unique to this model include: (i) interaction of the core region of the protein with the operator, (ii) primary effects of the conformational change in response to inducer on the core-operator interaction, (iii) contacts between all four subunits of the protein and the operator DNA, and (iv) qualitative differences in operator and nonspecific DNA binding.

Base Sequence↗
PubMedDate not supplied

Cigarette continuity programs and social support for smoking.

OBJECTIVE: To describe smokers' participation in cigarette continuity programs and the prevalence and structure of cooperative teams of smokers. METHODS: Cross-sectional survey of smoking histories and continuity-program participation by individuals and their family members in a convenience sample of 176 current smokers at the University of Kentucky Chandler Medical Center, Lexington. Fisher exact test or chi2 tests were used to compare proportions. RESULTS: One of 3 smokers collected coupons for a continuity program. Three quarters of the collectors redeemed their own coupons, and one quarter gave coupons to another collector. Coupon collectors reported an average team size of more than 2 members. One fifth of collectors were teammates with another generation of family members, and one quarter of collectors aged 24 to 35 years were teammates with their children. Smokers were often aware of their relatives' coupon-collecting habits. CONCLUSIONS: Continuity programs have been a popular means of reinforcing smoking, especially within families and groups of friends. Continuity programs are novel in encouraging smoking and brand loyalty between generations. Continuity-program participants need to be aware of the risk of promoting smoking initiation by their children. Health advocates could use similar strategies to promote smoking cessation and prevention within families and other social groups.

Adult↗