Limited functional redundancy and oscillation of cyclins in multinucleated Ashbya gossypii fungal cells.
ABSTRACT Cyclin protein behavior has not been systematically investigated in multinucleated cells with asynchronous mitoses. Cyclins are canonical oscillating cell cycle proteins, but it is unclear how fluctuating protein gradients can be established in multinucleated cells where nuclei in different stages of the division cycle share the cytoplasm. Previous work in A. gossypii, a filamentous fungus in which nuclei divide asynchronously in a common cytoplasm, demonstrated that one G1 and one B-type cyclin do not fluctuate in abundance across the division cycle. We have undertaken a comprehensive analysis of all G1 and B-type cyclins in A. gossypii to determine whether any of the cyclins show periodic abundance across the cell cycle and to examine whether cyclins exhibit functional redundancy in such a cellular environment. We localized all G1 and B-type cyclins and notably found that only AgClb5/6p varies in subcellular localization during the division cycle. AgClb5/6p is lost from nuclei at the meta-anaphase transition in a D-box-dependent manner. These data demonstrate that efficient nuclear autonomous protein degradation can occur within multinucleated cells residing in a common cytoplasm. We have shown that three of the five cyclins in A. gossypii are essential genes, indicating that there is minimal functional redundancy in this multinucleated system. In addition, we have identified a cyclin, AgClb3/4p, that is essential only for sporulation. We propose that the cohabitation of different cyclins in nuclei has led to enhanced substrate specificity and limited functional redundancy within classes of cyclins in multinucleated cells.
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ABSTRACT: Cyclin degradation is the key step governing exit from mitosis and progress into the next cell cycle. When a region in the N terminus of cyclin is fused to a foreign protein, it produces a hybrid protein susceptible to proteolysis at mitosis. During the course of degradation, both cyclin and the hybrid form conjugates with ubiquitin. The kinetic properties of the conjugates indicate that cyclin is degraded by ubiquitin-dependent proteolysis. Thus anaphase may be triggered by the recognition of cyclin by the ubiquitin-conjugating system.Nature 02/1991; 349(6305):132-8. · 36.28 Impact Factor
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ABSTRACT: Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.Journal of Molecular Biology 07/1983; 166(4):557-80. · 4.00 Impact Factor
Article: AgTHR4, a new selection marker for transformation of the filamentous fungus Ashbya gossypii, maps in a four-gene cluster that is conserved between A. gossypii and Saccharomyces cerevisiae.[show abstract] [hide abstract]
ABSTRACT: Single-read sequence analysis of the termini of eight randomly picked clones of Ashbya gossypii genomic DNA revealed seven sequences with homology to Saccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of the S. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putative AgTHR4 gene of A. gossypii. It comprises 512 codons, two less than the S. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of the A. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying the AgTHR4 gene and various S. cerevisiae ARS elements. Using these plasmids only very weak complementation of a S. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to the AgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved in A. gossypii and S. cerevisiae.MGG - Molecular and General Genetics 02/1996; 250(1):69-80.