@article {1210, title = {Modeling transcriptional regulation using microinjection into Xenopus oocytes.}, journal = {Methods}, volume = {17}, year = {1999}, month = {1999 Feb}, pages = {151-60}, abstract = {

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.

}, keywords = {Animals, Biological, DNA, DNA Primers, DNA-Binding Proteins, Fungal Proteins, Gene Expression Regulation, Genetic, Herpes Simplex Virus Protein Vmw65, Microinjections, Models, Oocytes, Recombinant Fusion Proteins, Saccharomyces cerevisiae Proteins, Transcription, Transcription Factors, Transcriptional Activation, Xenopus}, issn = {1046-2023}, doi = {10.1006/meth.1998.0726}, author = {Robinett, C C and Dunaway, M} } @article {1212, title = {The repeat organizer, a specialized insulator element within the intergenic spacer of the Xenopus rRNA genes.}, journal = {Molecular and Cellular Biology}, volume = {17}, year = {1997}, month = {1997 May}, pages = {2866-75}, abstract = {

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{\textquoteright} 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{\textquoteright}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.

}, keywords = {Animals, Genes, Genetic, Models, Molecular, Nucleic Acid, Repetitive Sequences, Reporter, Ribosomal, RNA, Sequence Deletion, Transcription, Transcription Factors, Xenopus laevis, Xenopus Proteins}, issn = {0270-7306}, author = {Robinett, C C and O{\textquoteright}Connor, A and Dunaway, M} } @article {1213, title = {GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion.}, journal = {Current Biology}, volume = {6}, year = {1996}, month = {1996 Dec 1}, pages = {1599-608}, abstract = {

Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy.

}, keywords = {Bacterial Proteins, Chromatids, Chromosomes, Escherichia coli Proteins, Fungal, Gene Expression, Green Fluorescent Proteins, Lac Repressors, Luminescent Proteins, Proteins, Recombinant Fusion Proteins, Repressor Proteins, Signal Transduction, Yeasts}, issn = {0960-9822}, author = {Straight, A F and Belmont, A S and Robinett, C C and Murray, A W} } @article {1214, title = {In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.}, journal = {The Journal of Cell Biology}, volume = {135}, year = {1996}, month = {1996 Dec}, pages = {1685-700}, abstract = {

We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.

}, keywords = {Anaphase, Animals, Base Sequence, CHO Cells, Chromatids, Chromatin, Chromosomes, Cricetinae, DNA, Electron, Gene Amplification, Gene Dosage, Green Fluorescent Proteins, In Situ Hybridization, Lac Operon, Luminescent Proteins, Microscopy, Mitosis, Molecular Sequence Data, Nucleic Acid, Recombinant Proteins, Repetitive Sequences, Staining and Labeling, Time Factors, Yeasts}, issn = {0021-9525}, author = {Robinett, C C and Straight, A and Li, G and Willhelm, C and Sudlow, G and Murray, A and Belmont, A S} }