Schizosaccharomyces pombe GATA factor Ams2 is responsible for cell cycle-dependent transcriptional activation of all the core histone genes peaking at G1/S phase. Intriguingly, its own protein level also fluctuates concurrently. Here, we show that Ams2 is ubiquitylated and degraded through the SCF (Skp1-Cdc53/Cullin-1-F-box) ubiquitin ligase, in which F box protein Pof3 binds this protein. Ams2 is phosphorylated at multiple sites, which is required for SCF(Pof3)-dependent proteolysis. Hsk1/Cdc7 kinase physically associates with and phosphorylates Ams2. Even mild overexpression of Ams2 induces constitutive histone expression and chromosome instability, and its toxicity is exaggerated when Hsk1 function is compromised. This is partly attributable to abnormal incorporation of canonical H3 into the central CENP-A/Cnp1-rich centromere, thereby reversing specific chromatin structures to apparently normal nucleosomes. We propose that Hsk1 plays a vital role during post S phase in genome stability via SCF(Pof3)-mediated degradation of Ams2, thereby maintaining centromere integrity.
This paper describes the production and characterization of a panel of 15 mouse monoclonal antibodies selected for putative activity against V-region related or allotypic determinants of a single IgG1 lambda paraprotein obtained from a patient with malignant lymphoplasmacytoid lymphoma. The specificity of each reagent for epitopes on the heavy (H) or light (L) chain or for conformational determinants (CD) of the immunogen was determined and the ability of one reagent to compete with another for these sites investigated. The fine specificity of the antibodies was assessed by screening on a large series of normal and paraprotein-containing sera. One monoclonal showed specificity for the Glm(f) allotype. The 14 other reagents identified a minimum of nine different epitopes in the V region of the immunogen, with four antibodies detecting private conformationally determined idiotypic specificities. Eight determinants were V-region markers also expressed on other paraproteins. A total of 30 out of 159 different paraproteins cross-reacted with one or more of the antibodies. Four of the shared epitopes were lambda-chain associated, three were H-chain associated and one was a conformational determinant. Homologies of lambda chain were identified more frequently among other paraproteins than those of H chain. The relationship between epitope expression and H-chain class of paraprotein was not random. The frequency of expression of cross-reactivities in association with IgG1 proteins was always exceeded by higher frequency of epitope expression in association with other classes of H-chain isotype. The potential therapeutic value of such panels of characterized monoclonal reagents is discussed.
Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division. Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed. Genetic analyses demonstrate that the sts5+ gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth. The sts5 mutant is not defective in cell wall integrity. Deletion of ppe1+, which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal. Multicopy plasmids containing either the protein kinase C-like gene pck1+ or the protein tyrosine phosphatase pyp1+, an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation. Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation. The predicted sts5+ gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis. The sts5+ gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants. Overexpression of the sts5+ gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed. Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting. A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure. These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.
The t(4;11)(q21;q23) has been associated with acute lymphocytic leukemia (ALL) especially in infants. The t(4;11) breakpoint on chromosome 11 is cytogenetically indistinguishable from breakpoints for other leukemia-associated translocations affecting 11q23. The molecular basis of the t(4;11) is unknown although a number of genes have been mapped to 11q23. The CD3D, G, and E genes have been positioned proximal to the 11q23 breakpoint of the 4;11 translocation while the THY1 and ETS1 genes have been mapped distal to this breakpoint. We report evidence that CD3G is within 200 kb of the 4;11 breakpoint as observed by pulsed field gel analysis. A rearrangement of the CD3G gene has been observed in a cell line derived from a patient with the t(4;11) translocation and in a hybrid cell line containing the derivative 11q chromosome derived from this cell line, using the restriction enzymes SacII and ClaI. Similar rearrangements using SacII were observed in 2 further patients with ALL and the t(4;11) translocation. No rearrangements in the same DNA were observed using ETS1, THY1, and D11S29 and a range of rare cutter restriction enzymes. CD3G thus provides a tool for the cloning and analysis of the 4;11 translocation, and poses a question of its possible involvement at long range with this translocation.
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