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M Plumpton

Publications and source records attributed to M Plumpton.

5 recordsLinked to original sources
PubMed1995

A high capacity assay for inhibitors of human papillomavirus DNA replication.

The discovery of antiviral compounds against human papillomaviruses (HPV) has been hindered by the difficulties in culturing virus in vitro or assaying stable HPV DNA replication. However, plasmids containing the HPV replication origin replicate transiently upon co-transfection with HPV E1 and E2 expression vectors. We have adapted this assay using secreted alkaline phosphatase (SAP) as a reporter for rapid analysis of DNA copy number. Use of the SV40 early promoter in controlling SAP expression was critical in ensuring both a strong signal and copy number dependence: the stronger beta-actin promotor inhibited replication, while the weaker SV40 late promoter yielded very low levels of SAP. The precise configuration of the E1 and E2 expression vectors also was critical, most pre-existing vectors did not support efficient replication and SAP secretion. The extent of DNA replication and SAP secretion were both proportional to the amount of E1/E2 vector used in transfections; under optimal conditions SAP increased 100-fold during replication. The assay has been developed for compound screening in 96-well plates and several inhibitors have been identified. Quantitative Southern blot analysis has shown that most of these inhibit HPV DNA replication rather than SAP accumulation or activity, and several are under test in models of viral replication. The assay also provides a rapid system for functional analysis of the HPV E1, E2 genes and the replication origin.

Alkaline Phosphatase↗
PubMed1994

A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes.

To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.

Base Sequence↗
PubMed1994

The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.

The RNA helicase-like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of interactions of PRP2 with spliceosomal components, PRP2 protein was stalled in splicing complexes by two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre-mRNAs, as demonstrated by UV-crosslinking experiments. The use of various mutant and truncated pre-mRNAs revealed that this interaction requires a spliceable pre-mRNA and an assembled spliceosome; a 3' splice site is not required. To extend these observations to the wild-type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre-mRNA in these spliceosomes in an ATP-independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S-ATP showed that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre-mRNA. As PRP2 is an RNA-stimulated ATPase, these experiments strongly suggest that the pre-mRNA is the native co-factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, we propose that the pre-mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing.

Adenosine Triphosphate↗
PubMed1992

The genetics of nuclear pre-mRNA splicing: a complex story.

The occurrence of introns in nuclear precursor RNAs (pre-mRNAs) is widespread in eukaryotes, and the splicing process that removes them is basically the same in yeasts as it is in higher eukaryotes. Splicing takes place in a very large, multi-component complex, the splicesome, and biochemical studies have been complicated by the large number of splicing factors involved. This review describes how genetic approaches used to study RNA splicing in Saccharomyces cerevisiae have complemented the biochemical studies and led to rapid advances in the field.

RNA Precursors↗