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Facile genetic and biochemical manipulation coupled with rapid cell growth and low cost of growth media has established the yeast Saccharomyces cerevisiae as a versatile workhorse. This article describes the use of yeast expression systems for the overproduction of complex multipolypeptide replication factors. The regulated overexpression of these factors in yeast provides for a readily accessible...
Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA...
Antisense RNA expression vectors have been developed relatively recently as a means to study the role of specific oncogenes in malignant transformation. In this paper, strategies for the construction of antisense plasmid vectors from commercially available reagents are described. Techniques for the introduction of these vectors into cell lines and tumors are also described and preferred methods for...
Recent work has demonstrated that the activity of a ubiquitous cellular enzyme, ribonuclease L (RNase L), can be harnessed to cleave targeted RNA species. Activation of RNase L is dependent on the presence of 2′,5′-linked oligoadenylates (2-5A), usually produced by cells infected with viruses. By conjugating synthetic 2-5A to specific antisense compounds, it is now possible to selectively degrade...
The mechanisms controlling duplication of the metazoan genome are only beginning to be understood. It is still unclear what organization of DNA sequences constitutes a chromosomal origin of DNA replication, and the regulation of origin activity during the cell cycle has not been fully revealed. We review recent results that indicate that chorion gene amplification in follicle cells of the Drosophila...
Small circular plasmids containing replication origins and, in some cases, centromeres, can replicate autonomously in the nuclei of all tested yeast species. Because this autonomous replication is dependent on the replication origin within the plasmid, measurements of the efficiency of autonomous replication (by the methods summarized here) permit evaluation of the effects of mutations on origin function...
Shotgun antisense is a technique to make a random set of mutant cells or organisms in such a way that one can select an interesting mutant and then sequence part of the mutated gene within a day. In addition to the fantastic rapidity with which one can identify the mutated gene, there are more advantages of this technique over other mutagenesis techniques: (1) one can identify genes that when completely...
Most researchers who intend to suppress a particular gene are interested primarily in the application of ribozyme technology rather than its mechanistic details. This article provides some background information and describes a straightforward strategy to generate and test a special design of a ribozyme: the asymmetric hammerhead ribozyme. This version of a hammerhead ribozyme carries at its 5′ end...
Short synthetic homopyrimidine- or homopurine-rich oligonucleotides can form sequence-specific triplexes with corresponding homopurine–homopyrimidine sites on duplex DNA and block transcription of a target gene in vitro. Such triplex-forming oligonucleotides (TFOs) can be rationally designed to target homopurine/homopyrimidine sequences that are often found in eukaryotic genes and thus used to modulate...
Schizosaccharomyces pombe has a cell cycle progression with distinctive phases that serves as a perfect model system for investigating DNA replication and repair of eukaryotic cells. Here, we use proliferating cell nuclear antigen (PCNA) of S. pombe to demonstrate how the function of this protein in both DNA replication and repair can be assessed by genetic and biochemical approaches. We describe...
Antisense transgenesis provides a methodology for ablating gene expression in targeted tissues through the use of tissue-specific or controllable promoters. The two major features to be considered in the design of a construct for injection are (1) the target sequence and (2) the promoter to be used. Information is provided to help the investigator make decisions in these regards. The standard methodology...
Antisense oligodeoxynucleotides (ODNs) are being explored as therapeutic agents for the treatment of many disorders including viral infections, cancers, and inflammatory disorders. In addition, antisense technology can be of great benefit to those attempting to assign function to the multitude of new genes being uncovered in the genomics initiative. However, the demonstration that the gene-regulating...
Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all levels of evolution. Is primary sequence, Drosophila melanogaster PCNA is 73% identical to mammalian PCNA. Moreover, it is able to substitute for mammalian PCNA in at least one intricate cell-free replication assay. Mutations in the gene for Drosophila PCNA,...
Two assays have been developed for studying DNA replication in vitro based on nuclear extracts isolated from budding yeast cells synchronized in S phase. In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G1. In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented...
Polyalkylcyanoacrylate nanoparticles are effective colloidal drug carriers and were prepared by an emulsion polymerization process. Antisense oligonucleotides were loaded on the particles by adsorption. A cationic polymer, DEAE-dextran, was incorporated into the particle matrix or a cationic hydrophobic detergent (CTAB) was used to form a lipophilic oligonucleotide ion pair. Enzymatic digestion of...
Experimental embryology has long used manipulation of interacting tissues to examine questions of tissue interaction and differentiation. The potential for specific manipulation of gene expression in such tissues has made the utilization of antisense techniques desirable. However, problems with this methodology have discouraged many investigators from using this approach. Selection of target sequences...
Minichromosome maintenance (mcm) is an effective genetic assay for mutants defective in DNA replication. Two classes of mcm mutants have been identified using this screen: those that differentially affect the activities of certain autonomously replicating sequences (ARSs) and those that uniformly affect the activities of all ARSs. The ARS-specific MCM genes are essential for the initiation of DNA...
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