Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis

Summary The cell division cycle gene CDC25 was replaced by various disrupted and deleted mutant copies. Mutants disrupted at a central position of the gene, or lacking 532 residues within the amono-terminal half of the gene product grow normally in glucose, but not in acetate media, and they fail to sporulate as homozygous diploids. Disruptions or deletions within the carboxy-terminal half are lethal, except for the deletion of the 38 carboxy-terminal residues, which are required for sporulation but not for growth in glucose or acetate media. It is concluded that distinct domains of the CDC25 gene product are involved in the control of mitosis and/or meiosis.     

Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis.

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.     

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.     

Ypi1, a positive regulator of nuclear protein phosphatase type 1 activity in Saccharomyces cerevisiae

The catalytic subunit of protein phosphatase type 1 (PP1) has an essential role in mitosis, acting in opposition to the Ipl1/Aurora B protein kinase to ensure proper kinetochore-microtubule interactions. However, the regulatory subunit(s) that completes the PP1 holoenzyme that functions in this capacity is not known. We show here that the budding yeast Ypi1 protein is a nuclear protein that functions with PP1 (Glc7) in this mitotic role. Depletion of cellular Ypi1 induces mitotic arrest due to activation of the spindle checkpoint. Ypi1 depletion is accompanied by a reduction of nuclear PP1 and by loss of nuclear Sds22, a Glc7 binding partner that is found in a ternary complex with Ypi1 and Glc7. Expression of a Ypi1 variant that binds weakly to PP1 also activates the spindle checkpoint and suppresses the temperature sensitivity of an ipl1-2 mutant. These results, together with genetic interactions among YPI1, GLC7, and SDS22 mutants, indicate that Ypi1 and Sds22 are positive regulators of the nuclear Glc7 activity that is required for mitosis.     

The F-box protein MAX2 functions as a positive regulator of photomorphogenesis in Arabidopsis

Light is vital for plant growth and development. To respond to ambient light signals, plants are equipped with an array of photoreceptors, including phytochromes that sense red (R)/far-R (FR) regions and cryptochromes and phototropins that respond to the ultraviolet-A/blue (B) region of the light spectrum, respectively. Several positively and negatively acting components in light-signaling pathways have been identified using genetic approaches; however, the pathways are not saturated. Here, we characterize a new mutant named pleiotropic photosignaling (pps), isolated from a genetic screen under continuous R light. pps has longer hypocotyls and slightly smaller cotyledons under continuous R, FR, and B light compared to that of the wild type. pps is also hyposensitive to both R and FR light-induced seed germination. Although photosynthetic marker genes are constitutively expressed in pps in the dark at high levels, the expression of early light-regulated genes is reduced in the pps seedlings compared to wild-type seedlings under R light. PPS encodes MAX2/ORE9 (for MORE AXILLARY BRANCHES2/ORESARA9), an F-box protein involved in inflorescence architecture and senescence. MAX2 is expressed ubiquitously in the seedling stage. However, its expression is restricted to vascular tissues and meristems at adult stages. MAX2 is also localized to the nucleus. As an F-box protein, MAX2 is predicted to be a component of the SCF (for SKP, Cullin, and F-box protein) complex involved in regulated proteolysis. These results suggest that SCF(MAX2) plays critical roles in R, FR, and B light-signaling pathways. In addition, MAX2 might regulate multiple targets at different developmental stages to optimize plant growth and development.     

A negative regulator of mitosis in Aspergillus is a putative membrane-spanning protein.

The temperature-sensitive cell cycle mutation bimE7 of Aspergillus nidulans causes cells to become blocked in mitosis at a restrictive temperature. Previous work has shown that this mitotic block is induced even when cells are arrested in the S or G2 phase. The mitotic block is also observed in cells carrying a null mutation in bimE, obtained by molecular disruption of the gene (Osmani, S.A., Engle, D.B., Doonan, J.H., and Morris, N.R. (1988) Cell 52, 241-251), indicating that a lack of bimE function is responsible for the phenotype. We have cloned the bimE gene by complementation of the mutant phenotype and have isolated and sequenced its corresponding cDNA. The gene product is encoded by a 6.5-7-kilobase mRNA. The deduced amino acid sequence suggests a protein with three transmembrane domains. The sequence contains numerous potential N-glycosylation sites and several putative cAMP-dependent phosphorylation sites. No homologous protein sequences were found in the common data bases. The bimE gene product is a novel component in the regulation of mitosis.     

Suppression of scant identifies Endos as a substrate of greatwall kinase and a negative regulator of protein phosphatase 2A in mitosis

Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwl(Scant), a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwl(Scant); many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo(11) partially sterile, even in the absence of gwl(Scant). Heterozygosity for an endos mutation suppresses this PP2A/polo(11) sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwl(Scant) dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop.     

Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.

Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function.     

A tobacco calcium/calmodulin-binding protein kinase functions as a negative regulator of flowering

A tobacco calcium/calmodulin-binding protein kinase (NtCBK1) was isolated and identified. The predicted NtCBK1 protein has 599 amino acids, an N-terminal kinase domain, and shares high homology with other calmodulin (CaM)-related kinases. Whereas NtCBK1 phosphorylates itself and substrates such as histone IIIS and syntide-2 in the absence of CaM, its kinase activity can be stimulated by tobacco CaMs. However, unlike another tobacco protein kinase designated NtCBK2, NtCBK1 was not differentially regulated by the different CaM isoforms tested. The CaM-binding domain of NtCBK1 was located between amino acids 436 and 455, and this domain was shown to be necessary for CaM modulation of kinase activity. RNA in situ hybridization showed that NtCBK1 was highly regulated in the transition to flowering. Whereas NtCBK1 mRNA was accumulated in the shoot apical meristem during vegetative growth, its expression was dramatically decreased in the shoot apical meristem after floral determination, and in young flower primordia. The expression of NtCBK1 was up-regulated to high levels in floral organ primordia. Fluctuations in NtCBK1 expression were verified by analysis of tobacco plants expressing green fluorescent protein under the control of the NtCBK1 promoter, suggesting a role of NtCBK1 in the transition to flowering. This conclusion was confirmed by overexpressing NtCBK1 in transgenic tobacco plants, where maintenance of high levels of NtCBK1 in the shoot apical meristem delayed the switch to flowering and extended the vegetative phase of growth. Further work indicated that overexpression of NtCBK1 in transgenic tobacco did not affect the expression of NFL, a tobacco homologue of the LFY gene that controls meristem initiation and floral structure in tobacco. In addition, the promotion of tobacco flowering time by DNA demethylation cannot be blocked by the overexpression of NtCBK1.     

Beta1,4-galactosyltransferase V functions as a positive growth regulator in glioma

beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAcbeta1-->6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.     

A transcriptional cascade governs entry into meiosis in Saccharomyces cerevisiae.

Two signals activate meiosis in yeast: starvation and expression of the a1 and alpha 2 products of the mating-type locus. Prior studies suggest that these signals stimulate expression of an activator of meiosis, the IME1 (inducer of meiosis) product. We have cloned a gene, IME2, with properties similar to those of IME1: both genes are required for meiosis, and both RNAs are induced in meiotic cells. Elevated dosage of IME1 or IME2 stimulates the meiotic recombination pathway without starvation; thus, the IME products may be part of the switch that activates meiosis. IME1 was found to be required for IME2 expression, and a multicopy IME2 plasmid permitted meiosis in an ime1 deletion mutant. Accordingly, we propose that the IME1 product stimulates meiosis mainly through activation of IME2 expression.