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Title: Cytological, biochemical and molecular analysis of mitotic control proteins in higher plants
Author: Young, Tania
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1992
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Probes derived from cell cycle genes and proteins of the fission yeast Schizosaccharomyces pombe were used to investigate cell cycle control mechanisms in higher plants at the biochemical, cellular and molecular biological level. Full-length cdc2+, cdc13+ and suc1+ genes were used to screen Southern blots of genomic DNA from different plant species. The cdc13+ gene hybridised to Chlamydomonas, maize and Arabidopsis DNA but yielded no positives when used to screen an Antirrhinum cDNA library. Two oligonucleotides were designed, complementary to regions conserved in cdcl3 and its homologues, with the intention of screening a second cDNA library made from Arabidopsis buds. The oligonucleotides were also used in the polymerase chain reaction (PCR) with maize genomic DNA. A 'ladder' of DNA fragments around the expected size was amplified. Polyclonal antibodies against yeast cell cycle proteins were used for indirect immunofluorescence staining of onion root tip cells. An antibody against the conserved PSTAIR epitope of the cdc2+ gene product stained a cytoplasmic reticulum but this staining was not abolished by competition with PSTAIR peptide. The monclonal antibody MPM2 recognised a nuclear antigen which became dispersed throughout the cytoplasm at mitosis. Nuclear staining was brightest at prophase and, by double staining for tubulin, this stage was always found to correlate with the presence of a preprophase band of microtubules. p13, the suc1+ gene product, was purified from a bacterial expression system and coupled to Sepharose. p13-Sepharose precipitated a histone H1 kinase activity and a protein possessing the PSTAIR epitope from extracts of tobacco suspension cells released from anaphidicolin block. The precipitated kinase activity did not vary during the cell cycle of these synchronised cells. p13-Sepharose also precipitated a histone H1 kinase activity from an extract of carrot suspension cells in stationary phase but no protein possessing the PSTAIR epitope was detected in this case.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available