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Phase-locking (frequency entrainment) of an oscillator, in which a periodic extrinsic signal drives oscillations at a frequency different from the unperturbed frequency, is a useful property for study of oscillator stability and structure. The cell cycle is frequently described as a biochemical oscillator; however, because this oscillator is tied to key biological events such as DNA replication and segregation, and to cell growth (cell mass increase), it is unclear whether phase locking is possible for the cell cycle oscillator. We found that forced periodic expression of the [G.sub.1] cyclin CLN2 phase locks the cell cycle of budding yeast over a range of extrinsic periods in an exponentially growing monolayer culture. We characterize the behavior of cells in a pedigree using a return map to determine the efficiency of entrainment to the externally controlled pulse. We quantify differences between mothers and daughters and how synchronization of an expanding population differs from synchronization of a single oscillator. Mothers only lock intermittently whereas daughters lock completely and in a different period range than mothers. We can explain quantitative features of phase locking in both cell types with an analytically solvable model based on cell size control and how mass is partitioned between mother and daughter cells. A key prediction of this model is that size control can occur not only in [G.sub.1], but also later in the cell cycle under the appropriate conditions; this prediction is confirmed in our experimental data. Our results provide quantitative insight into how cell size is integrated with the cell cycle oscillator. cell size control | phase locking

Is there a cellular mechanism for preventing a depolymerizing microtubule track from 'slipping out from under' its cargo? A recent study in budding yeast indicates that when a chromosome is transported to the minus end of a spindle microtubule, its kinetochore-bound microtubule plus end-tracking protein (+TIP) Stu2 may move to the plus end to promote rescue; i.e., to switch the depolymerizing end to a polymerizing end. The possibility that other +TIPs may play a similar role in sustaining a microtubule track during vesicular transport deserves investigation.

ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1(delta) synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2(delta) mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-(alpha)-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2(delta) cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

Retinoic acid-treated mesenchyme cells of the budding ascidian Polyandrocarpa misakiensis acquire an organizer activity to induce a secondary body axis when implanted into developing buds. We identified several different mRNAs that were upregulated in the mesenchyme cells after retinoic acid treatment. We isolated a cDNA clone corresponding to one of these mRNAs. The C-terminal region of the predicted protein product is homologous to the catalytic domain of serine proteases that belong to the trypsin family. The N-terminal region contains several types of protein-protein interaction domains. We therefore named this protein tunicate retinoic acid-inducible modular protease (TRAMP). Expression of the TRAMP mRNA in mesenchyme cells during budding and its upregulation by retinoic acid were demonstrated by reverse transcription-PCR and in situ hybridization. A glutathione S-transferase-TRAMP fusion protein showed a protease activity with trypsin-like substrate specificity and stimulated proliferation of the cell line established in this species.

The yeast Saccharomyces cerevisiae forms a ring of chitin in its cell wall prior to bud emergence. The chitin ring consist mainly of chitin synthase III. In experiments conducted, it has been observed that both the absence of the gene BNI4 and overexpression of Bni4p lead to the delocalization of chitin deposition and aberrant cellular morphology. Localization depends on the presence of four septin proteins but not on Chs4p or Chs3p. These findings suggest the link between Chs3p and the septins via Chs4p and Bni4p during chitin synthase III localization.

A study was conducted on the Candida albicans RAS-related gene (CaRSR1), which was isolated as a suppressor of a cdc24 (sup ts) bud-emergence mutation of Saacharomyces cerevisiae. Results have shown that (CaRSR1) is needed to limit yeast budding to the cell poles and is involved in germ tube emergence, hypha growth and cell morphogenesis. Moreover, the virulence of the pathogen is significantly affected by the disruption of (CaRSR1).

Lithium ions affect pattern formation in the freshwater polyp Hydra vulgaris in a complex manner. Although a long-term treatment with 1 mM LiCl completely suppresses both head formation and budding, a reduction of the lithium concentration during a long-term treatment from 4 to 1 mM LiCl strongly promotes the differentiation of ectopic head structures. Meanwhile, budding, often interpreted as a special case of head formation, remains suppressed. The appearance of ectopic tentacles under these conditions is surprising as they develop while the animals remain in the presence of 1 mM LiCl, a concentration which would normally suppress the formation of tentacles. On a molecular level, the induction of ectopic head structures by the shift in the LiCl concentration is preceded by an increase in the level of inositol phosphates, indicating an activation of the phosphatidylinositol (PI) cycle. Increased inositol phosphate levels persist for at least 24 hr. Our results provide evidence that (i) tentacle- and bud-forming systems need different and possibly mutually exclusive physiological preconditions and (ii) prolonged inhibition of the PI cycle after initial activation might be necessary to obtain ectopic tentacles.

The Bud4 protein in the yeast, Bud4p, is essential for the axial budding pattern of cells and is localized in the mother/bud neck, which is the site of the neck filaments. Bud4p rings exist in cells after cytokinesis, but disappear before buds emerge. Axial budding is facilitated through the cooperation of Bud4p and the Bud3 protein. The 2 proteins are involved in a cycle of protein-protein interactions which marks the bud site.

The second src homology 3 (SH3) region from the NH2 terminus of the Bem1p is important bud formation and suppression of rho3 defect. Boi2p found bound to SH3-2 has a proline-rich region that is critical for displaying Boi2p-Bem1p two-hybrid reaction. THe COOH-terminal is essential for the function of BOI2p in cell growth, while the NH2-terminal is requires for the modulating the function of Bem1p. Rho3p GTPases and Boi proteins function in the maintenance of cell polarity for bud formation.

Research into the cell division of Saccharomyces showed cells forming the septin ring to define the cleavage plane as an eventual budding site. The septin ring acts as a gate where the dividing nucleus, the forming actin and microtubule cytoskeleton aggregate prior to their migration to the forming bud. The same septin ring divides as the cell divides and disintegrates as soon as the budding units have separated.

The BED1 gene of Saccharomyces cerevisiae is essential for polarized growth and efficient budding. BED1 encodes an alpha-1,2-galactosyltransferase homologue, an integral membrane protein present in the endoplasmic reticulum. The BED1 mutant cell neither exhibits bud emergence nor possesses the ellipsoidal shape specific for wild-types, and are bigger than the wild-type cells. An intact morphogenesis checkpoint control helps in the survival of the mutants. Polarized growth and bud emergence depend on the glycosylation of specific proteins or lipids.

Immunolocalization and genetic observation of budding yeast shows that Bud3p assembles in the mother-bud before nuclear division and cytokinesis by binding to the neck filaments or their associated proteins. After division, it remains localized as a single ring on the surface of mother and daughter cells. Bud3p and neck filament loose their localization on shifting the cdcl2 mutant to restrictive temperature.

Research into budding yeast reveals that the division site and the distal tip of the cell carry the axial and the bipolar spatial signals which direct the selection of bud site in the bipolar cell cycle. In the axial pattern, the new bud forms adjacent to the preceding division site in mother cells and adjacent to the division site in daughter cells. Refeeding and starvation of axially budding cells produce new buds at nonaxial sites.

In saccaromyces cerevisae, the delaying of the cell cycle in Cdc24 and other mutants by phosphorylation of tyrosine 19 of Cdc28 indicates the presence of cell morphogenesis mediating checkpoint in budding yeast. The phosphorylation of tyrosine 19 of Cdc28 inhibits Cdc28 activation, and the delay in nuclear division caused by the inhibition of bud formation permits the cell to recoup from short term disruption of cell polarity without becoming binucleate.

Early events of multiple bud formation and shoot development in germinating soybean embryonic axes treated for 24 hr with the cytokinin, 6-benzylaminopurine (BAP), were compared to the development of untreated control axes using four different techniques: photomicrography, scanning electron microscopy, histology, and autoradiography. Shoot apex development in BAP-treated embryonic axes was delayed by about 9 to 15 hr. A transient inhibition of DNA synthesis in the primary apical meristem and axillary buds was observed with subsequent changes in the timing of cell division patterns in these regions. Meristematic regions (supernumerary vegetative buds) were observed in BAP-treated axes around the perimeter of the apical dome at and above the level of the axillary buds. Cells elongated from some of the BAP-induced meristematic regions to form four to six shoots. In the absence of BAP, excision of the primary apical meristem and/or axillary buds did not result in multiple bud formation. These results suggest that transient exposure to BAP interrupted chromosomal DNA replication and reprogrammed the developmental fate of a large number of cells in the shoot apex. We postulate that interruption of DNA synthesis, either directly, by interfering with DNA replication, or indirectly, by preventing entry into S-phase, effected redetermination of the shoot apex cells.

Intracellular transfers between membrane-bound compartments occur through vesicles that bud from a donor compartment to fuse subsequently with an acceptor membrane. We report that the membrane that delimits COP I or COP II-coated buds/vesicles from the endoplasmic reticulum and the Golgi complex has a thinner interleaf let clear space as compared with the surrounding, noncoated parental membrane. This change is compatible with a compositional change of the membrane bilayer during the budding process.

Budding in the genus Hydra was first described in the scientific literature, and sketched, by Leeuwenhoek (1). Since that time, budding has continued to be a popular problem for developmental biologists who study Hydra (2, 3, 4). The relationship between feeding and budding was established in mass culture experiments performed by a number of investigators (5, 6, 7, 8, 9). The discovery by Schaller (10) of a low-molecular-weight peptide released by the nerve cells of Hydra in response to feeding or injury, and her determination that this polypeptide acts as a mitogen (11), taken together with studies showing a correlation between the mitotic index and the budding rate in Hydra (7, 12, 13) left little doubt that feeding and budding are causally related. Recent studies continue to reinforce the observation that reduced glutathione (GSH), or glutathione derivatives, can elicit the feeding response in Hydra (14, 15). But the literature is mute about the possibility that GSH, which causes a mechanical feeding response in Hydra (16), might also stimulate budding. This study was designed to test that possibility. The specific objectives were (a) to determine whether exposure to GSH leads to a significant increase in the rate of bud initiation in starved Hydra oligactis; (b) to determine by employing decapitated H. oligactis, whether any observable GSH effect can occur without mediation by the head of the hydra; and (c) to determine whether any GSH induction of budding is additive to that elicited by feeding. All of these objectives were met, and we propose that GSH plays a (significant) role in the cascade of events heading to budding in Hydra.

A meiosis-specific protein (Hed1) with a novel Rad51 regulatory function is described. It is proposed that Hed1 protein provides a mechanism to ensure the coordinated action of Rad51 and Dmc1 during meiosis, by down-regulating Rad51 activity when Dmc1 is unavailable.

The molecular mechanisms involved in selective cargo transport between organelles are studied. The transport by vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment is discussed.

Many intracellular compartments of eukaryotic cells do not adopt a spherical shape, which would be expected in the absence of mechanisms organizing their structure. However, little is known about the principles determining the shape of organelles. We have observed very defined structural changes of vacuoles, the lysosome equivalents of yeast. The vacuolar membrane can form a large tubular invagination from which vesicles bud off into the lumen of the organelle. Formation of the tube is regulated via the Apg/Aut pathway. Its lumen is continuous with the cytosol, making this inverse budding reaction equivalent to microautophagocytosis. The tube is highly dynamic, often branched, and defined by a sharp kink of the vacuolar membrane at the site of invagination. The tube is formed by vacuoles in an autonomous fashion. It persists after vacuole isolation and, therefore, is independent of surrounding cytoskeleton. There is a striking lateral heterogeneity along the tube, with a high density of transmembrane particles at the base and a smooth zone devoid of transmembrane particles at the tip where budding occurs. We postulate a lateral sorting mechanism along the tube that mediates a depletion of large transmembrane proteins at the tip and results in the inverse budding of lipid-rich vesicles into the lumen of the organelle. Key words: microautophagocytosis * lysosome * yeast * proteolysis * budding

The genetic control of bud phenology in hybrid poplar was studied by mapping quantitative trait loci (QTL) affecting the timing of autumn bud set and spring bud flush. The founders of the mapping pedigree were collected from widely separated latitudes to maximize segregating variation for dormancy-related traits in the [F.sub.2] generation--the female Populus trichocarpa parent is from Washington State (48 [degrees] N) and the male P. deltoides parent is from Texas (31 [degrees] N). Bud set and bud flush timing were measured on the [F.sub.2] generation in a replicated clonal field trial. Using a linkage map constructed of AFLP and microsatellite markers, three QTL controlling bud set and six QTL controlling bud flush were detected. Additionally, five candidate genes believed to be involved in perception of photoperiod (PHYB1, PHYB2) or transduction of abscisic acid response signals (ABI1B, ABI1D, and ABI3) were placed on the QTL map. PHYB2 and ABI1B were found to be coincident with QTL affecting bud set and bud flush.

Cells of the yeast Saccharomyces cerevisiae have seven protein mannosyl transferases (pmt) that are needed to catalyze the initial step of O-linked glycosylation. These enzymes, which are also involved in cell morphology and cell wall integrity, are shown to be involved in the nuclear and morphological cell cycles. Specifically, it is shown that O-glycolysation of Axl2/Bud10p by pmt4 is needed for the stability, localization and function of daughter cells.

In the freshwater coelenterate, hydra, asexual reproduction via budding occurs at the base of the gastric region about two-thirds of the distance from the head to the foot. Developmental gradients of head and foot activation and inhibition originating from these organizing centers have long been assumed to control budding in hydra. Much has been learned over the years about these developmental gradients and axial pattern formation, and in particular, the inhibitory influence of the head on budding is well documented. However, understanding of the role of the foot and potential interactions between the foot, bud, and head patterning systems is lacking. The purpose of this study was to investigate the role of the foot in the initiation of new axis formation during budding by manipulating the foot and monitoring effects on the onset of first bud evagination and the time necessary to reach the 50% budding point. Several experimental situations were examined: the lower peduncle and foot (PF) were injured or removed, a second PF was laterally grafted onto animals either basally (below the budding zone) or apically (above the budding zone), or both the head and PF were removed simultaneously. When the PF was injured or removed, the onset of first bud evagination was delayed and/or the time until the 50% budding point was reached was longer. The effects were more pronounced when the manipulation was performed closer to the anticipated onset of budding. When PF tissue was doubled, precocious bud evagination was induced, regardless of graft location. Removal of the PF at the same time as decapitation reduced the inductive effect of decapitation on bud evagination. These results are discussed in light of potential signals from the foot or interactions between the foot and head patterning systems that might influence bud axis initiation. Key Words: hydra; pattern formation; budding; axis induction; gradients.

The relationship between the yeast centrin Cdc31p and the interacting protein kinase Kic1p is examined. A two-hybrid screen performed for Cdc31p-interacting proteins identified the protein kinase Kic1p that phosphorylates substrates. The gene is described as critical for cell integrity or morphogenesis although it is not applicable to SPB duplication. Moreover, cdc31 mutants also influence cell integrity and morphogenesis. These findings suggest that Cdc31p functions via Kic1p to control cell integrity or morphogenesis.