{'fraction_non_alphanumeric': 0.03507820969490476, 'fraction_numerical': 0.024650250374270818, 'mean_word_length': 4.841827503015682, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDC10, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. Biol. 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normal-looking ring structures at the bases of the first zygotic buds.

Abstract. Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDCIO, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. BioL 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies deco-rated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normallooking ring structures at the bases of the first zygotic buds.
T HE cell cycle of the yeast Saccharomyces cerevisiae involves a series of morphogenetic events including highly localized changes in the cell surface (7,34,40). As in other eukaryotes, elements of the cytoskeleton are believed to play key roles in these morphogenetic events. In addition to microtubules (2,4,18,19,21) and actin-containing structures (2,21,30), yeast cells contain another cytoskeletal element, of uncertain biochemical nature, that may be involved in morphogenesis. This is a highly ordered array of filaments, •10 nm in diameter, that lies immediately subjacent to the cellular membrane in the region of the motherbud neck (4,5). By EM, these filaments seem to appear at the onset of bud emergence and to disappear just before cytokinesis. Temperature-sensitive mutants defective in any of the CDC12 product. In addition, we have used the CDC3specific and CDC12-specific antibodies to investigate the timing of filament assembly and disassembly relative to other landmark events at the beginning and end of the cell cycle. We have also used the antibodies to ask whether structures similar to the filament ring of budding cells appear during the morphogenetic response to mating pheromone (9,26,36,41,42), during zygote formation (28,41,43), and during formation of the first zygotic bud.

Plasmids, Strains, and Growth Conditions
Plasmid pUR290 (35) and E. coli strain BMH 71-18 (29) were provided by R. Schekman and were handled essentially as described previously (35). Plasmid pATH2 was provided by T. Koerner and A. Tzagoloff and was propagated in E. coli strain I-IBI01 (27) essentially as described previously (10). Other plasmids are described in reference 25 or in Results.
The yeast strains used are listed in Table I. Except as noted, these were grown with rotary shaking in the rich, glucose-containing medium YM-P (24) at "023"C. Strain TD4 was pmpegated in the appropriate selective minimal media (10) either without plasmid or after transformation with appropriate plasmids. Temperature-sensitive cell-division-cycle mutant strains were grown to 'o107 cells/ml at "023"C, then shifted to 36"C either without dilution or by diluting sixfold with fresh, prewarrned medium.

Alpha-Factor Arrest, Mating, and Synchronization
To arrest cell proliferation with alpha factor, strain CP1AB-1AA was grown to "0107 cells/ml, then diluted in fresh medium to a density of 2 × 106 cells/ml. Alpha factor was then added (from a 1-mg/ml stock in water) to a final concentration of 5 #g/ml and incubation was continued. For mating experiments, strains C276-4A and C276-4B were grown to "0107 cells/mi. Equal numbers of cells of opposite mating types were mixed and collected on 0.45-#m filters to yield 4 x 10 ~ to 4 x 107 cells per 2.5-cm diameter filter. The filters were placed on YEPD plates and cells were collected after various periods of incubation by washing a filter with two 5-ml changes of phosphate buffer and adding formaldehyde to a final concentration of 3.7 %. Alternatively, the haploid strains were grown to "00.5 × 107 cells/ml, cultures were mixed so as to provide equal numbers of cells of each mating type, and incubation was continued with slow rotary shaking.
Two methods were used to obtain partially synchronized populations of budding cells. In the first, strain C276 cells were grown to early stationary phase and small cells were isolated using a Ficoll (mol wt 400,000) stepdensity gradient. 2 mi of culture were layered on a gradient of 4, 5, 6, and 7% Ficoll in sterile water (32 mi in a 50-ml centrifuge tube) and spun in a Sorvall GLC-2B tabletop centrifuge for 5 rain at 800 rpm at "023"C. Approximately 3 ml from the top of the gradient were removed and the cells in this fraction were harvested by spinning for 10 min at 1,500 rpm. These cells were predominantly unbudded and bud scar free (as judged by Caicofluor staining; 10, 40). The cells were washed with 10 mi of YM-P medium to remove the remaining Ficoll and were then resuspended and incubated in 10 ml of fresh medium. In the second method, cells exposed to alpha factor for 160 rain as described above were harvested by centrifugation at "023"C, then resuspended and incubated in an equal volume of fresh medium.

Preparation of Antigens and Antibodies
The lacZ:CDC3 fusion protein was prepared from strain BMH 71-18 hat-  Fig. 1 B). Although this plasmid retains cdc3-complementing activity, sequence analysis suggests that it does not contain the entire CDC3 c o~g region (11, 25; Fig. 1, A and B; and see below). To create a IacZ:CDC3 fusion gene, the 1.3-kbp Pvu II Hind HI fragment from YEp-3PS1 was inserted into plasmid pUR290 (35) to produce plasmid pUR-PH1 as shown in Fig. 1 C. Analysis of the CDC3 sequence 01) indicated that this arrangement v, vuld result in an inframe fusion of the/acZ and CDC3 genes. Indeed, pUR-PH1 directed the synthesis ofa ,,ol60-kD fusion protein (Fig. 2 A,  lanes 1-3) when propagated under inducing conditions. A trpE:CDC3 fusion gene was constructed by inserting the same Pvu H-Hind HI fragment into plasmid pATH2 to produce plasmid pATH-PH8 as shown in Fig. 1 C. This plasmid boring fusion plasmid pUR-PH1 (see Results). Cells were grown overnight in LB medium plus ampicillin (50/tg/ml) at 37°C, then diluted 1:10 in the same medium and grown 1 h at 37°C. Isopropyl ~-v-thiogalactopyranoside (25 mg/rul in H20) was added to a final concentration of ~/ t g / m l , and cultures were grown for two more hours at 37°C. Cells were then harvested, resuspended in loading buffer (10), and placed in a boiling water bath for 1 to 2 min before loading samples on a pulyacrylamide gel (10). The trp E: CDC3 fusion protein was prepared from E. coil swain HB101 containing plasmid pATH-PH8 (see Results). Induction of the fusion protein and isolation of an insoluble protein fraction were essentially as described previously (10) for the trpE:CDC12 fusion proteins. For immunization, both CDC3 fusion proteins were purified from polyacrylamide gel slices and injected separately into rabbits as described previously (10).

Indirect Immunofluorescence and Other Methods
Cells were ordinarily fixed by the addition of 37 % formaldehyde directly to the culture medium (to a final concentration of 3.7%), then examined by phase-contrast microscopy (32) or washed and prepared for indirect immunofluorescence as described previously (2,10,21). Doubie-label experiments used FITC-conjugated goat anti-rabbit IgG to visualize rabbit anti-CDC3 or anti-CDCl2 antibodies, plus rhodamine-conjugeted goat anti-rat IgG to visualize rat antitubulin antibodies. Chitin rings were visualized by staining with Calcofluor as described previously (10,40). Other methods were as described previously (10,32).

R e s u l t s Construction o f lacZ : C D C 3 and trp E : C D C 3 Gene Fusions
From the CDC3-contairtng plasmid YEpl3(CDC3)I, a 2.4kbp Pvu II-Sca I fragment was subeloned into the yeast-E. coli shuttle vector YEpl02 to create plasmid YEp-3PS1

Preparation of CDC3-specific Antibodies and Identification of the CDC3 Protein
The lacZ: CDC3 and trpE: CDC3 fusion proteins were used to elicit antibody production and the resulting antisera were tested by immunoblotting for their ability to recognize proteins from the appropriate E. coli strains. We obtained sera raised against each fusion protein that were able to recognize both fusion proteins in Western blots (data not shown; see similar experiments with anfisera raised against CDC12 fusion proteins in reference 10). Thus, each serum appeared to contain antibodies specific for CDC3 determinants that are common to the two fusion proteins.
The Journal of Cell Biology, Volume 112, 1991 The CDC3-specific antibodies were affinity purified using nitrocellulose-bound fusion proteins and the purified antibodies were used to identify the authentic CDC3 product in blots of yeast proteins. A "062-kD protein was recognized in blots of total protein from CDC3 + cells containing no plasmid (data not shown) or plasmid YEP102 (Fig. 2 B, lane/). A protein of the same mobility was present at higher levels in extracts of the same strain harboring a high-copy-number plasmid containing the entire CDC3 coding region (Fig. 2  B, lane 3), indicating that the "062-kD protein is the authentic CDC3 gene product. In addition, a plasmid containing a tnmcated CDC3 coding region in the same strain directs the synthesis of a "050-kD protein (Fig. 2 B, lane 2; see Discussion).

Immunofluorescence Localization of the CDC3 Gene Product
Use of affinity-purified anti-trpE:CDC3 (Fig. 3 A) or anti-lacZ:CDC3 (data not shown) antibodies to stain wild-type yeast cells revealed a localization pattern very similar to that seen for the CDCI2 gene product (10). In cells with small buds, the CDC3 gene product appeared in a single or double ring at the base of the bud (Fig. 3 A, cells a-c). In cells with medium or large buds, the CDC3 gene product appeared as a pair of rings flanking the constriction of the mother-bud neck ( Fig. 3 A, cells d-f). As noted previously (10), this appearance is that expected for an antigen present in a structure, such as the ring of 10-nm filaments, that follows the contour of the cell membrane through the neck region; thus, densest packing (in a two-dimensional projection), and therefore brightest fluorescence, would occur on the slopes of the mother and bud portions of the cell flanking the neck constriction. Finally, many unbudded cells also appeared to possess rings of CDC3 antigen (Fig. 3 A, cells g-l), as considered further below.
Mutants defective in CDC3, CDC]O, CDCll, or CDC12 are unable to form or (at least for some alleles of CDCIO and CDC12) maintain rings of 10-nm filaments at restrictive temperature (Byers, B., and L. Goetsch. 1976. J. Cell Biol. 70:35a; 1, 10). The pattern of CDC3 product localization was examined in representative mutant strains. At permissive temperature, all four mutants displayed normal localization of the CDC3 product (Fig. 3, B, D, and F), although the staining was somewhat diminished in cdc3 (Fig. 3 B) and cddl (strain JPT194-H01; data not shown) cells. After 30 rain at restrictive temperature (sufficient to cause disappearance of 10-nm filaments [1; Byers, B., and L. Goetsch. 1976. J. Cell Biol. 70:35a] and delocalization of CDC12 product [10] in the cddO and cdcl2 cells), staining of CDC3 product was not detectable in cdc10 and cdcl2 cells (Fig. 3, E and G) and was greatly diminished or absent in cdc3 cells (Fig.  3 C). cdcll cells showed only a minor reduction in staining intensity relative to the pattern observed at permissive temperature (data not shown). Moreover, a cdc4 mutant strain (which also forms multiple, abnormally elongated buds at restrictive temperature, but has normal rings of 10-nm illaments) displayed apparently normal localization of CDC3 product to the mother-bud necks even after several hours at restrictive temperature (Fig. 3 H). Thus, the results obtained with the mutant strains are also consistent with the hypothesis that the CDC3 product is a constituent of the ring of 10nm filaments.
Electron microscopic observations have suggested that the ring of 10-nm filaments appears coincident with bud emergence and disappears just before cytokinesis (4,5). To begin exploring the behavior of the filament-associated polypeptides at the beginning and end of the budding cycle, we examined in detail the patterns of staining observed with CDC3specific and CDC12-specific antibodies in exponentially growing wild-type cells. In a sample of strainC276, 62% of the cells were budded as judged by phase-contrast microscopy. Immunofluorescence revealed detectable rings of the CDC3 and CDC12 antigens in 96 and 80% of the total population, respectively. When we attempted to score the unbudded cells alone, we detected rings of CDC3 and CDC12 product in 93 and 40% of the ceils, respectively.-However, it should be noted that the latter counts are somewhat uncertain because small buds are difficult to see under the conditions used for immunofluorescence; thus, cells with small buds may be inadvertantly included in the counts, with the effect of inflating the apparent proportions of unbudded cells that possess rings of CDC3 or CDC12 antigen. Nonetheless, it seems clear that both antigens (and especially the CDC3 product) must (a) be organized into a ring structure before bud emergence; (b) linger in a ring structure after cytokinesis; or (c) both. Support for possibility c is provided by the observation that the rings of antigen observed in unbudded cells appear to be of two types: first, a relatively narrow diameter, generally brightly staining structure (Fig. 3 A, cells g, h, and k [right-hand ring], and l [left-hand ring]; Fig. 3  I, cells a and b), such as would be expected if the structures observed at the bases of small buds (Fig. 3 A, cells a-c; Fig.  3, D and E of ref. 10) actually appeared in advance of bud emergence; and second, a relatively broad-diameter, generally less brightly staining structure (Fig. 3 A, Fig. 3 A, were retained on mother and daughter cells after cytokinesis. The narrow and broad rings were present in "035 and "058%, respectively, of the apparently unbudded cells stained with CDC3-specific antibodies, and were each present in ,020% of the apparently unbudded cells stained with CDC12-specific antibodies. In addition, a small number of apparently unbudded cell s (and even some cells with small buds) displayed coincidentiy both a narrow ring and a broad ring when stained with CDC3-specific antibodies (Fig. 3   ceils a, c, k, and 1), providing further support for possibility c, above. However, cells with two rings were not observed after staining with CDC12-specific antibodies.

Timing of Early Events at the Budding Site
To examine more closely the timing of the appearance of the CDC3 and CDC12 proteins at the budding site, small unbudded cells were isolated and cultured in fresh mediuna. The numbers of buds, chiti~ rings, and tings of CDC3 or CDC12 antagen were then compared during the partaally synchronous round of budding that follo~;ved (Fig. 4 A). Theini-ti~ ce'll population eQntained some cells with bud scars (i:e., chidfi tings from previous budding cycles) but few or none ~vith'bfi~ls or with detectable tings of CDC3 or CD02 antigen..~us, at each tiiiie point, the total number of chitin tings is somewhat greater than the number of newly formed chitin tings, whereas the buds and antigen tings observed are pFesumably all newly formed. With this in mind, Comparison of the curves in Fig. 4 A suggests'that the CDC3 and CDC12 proteins localize to the budding site before bud eme rgence and apparently also before chitin ring formation. It shduld ~ noted, however, that immunofluorescende ~taining in the egrlier samples (up to 75 rain) seemed suboptimal, pres0mably because of difficulties in permeabilizing cells that were just emerging from stationary phase. Therefore, the co.~nts of antigen rings in these early samples may have been distorted by a failure to detect some residual rings from earlier bl~dding cycles, nascent tings from the new budding cycles,' or both. Because of this caveat, we also utilized a second method to synchronize budding. MATa/MATa diploid cells Were treated with alpha factor to arrest the cells uniformly ~ the unbudded, G1 phase (33). After washing ihe cells and resuspending them in fresh medium, the numbers of buds and tings of CDC3 or CDC12 antigen were determined a s a function of time. (The formation of new chitin rings could not be followed in this experiment because of the many bud scars present in the initial population.) Fig. 4 B shows that the rings of CDC3 antigen appeared to form ,,o15 .min before bud emergence, whereas the rings of CDC12 antigen appeared to form approximately coincident with bud emergence. The apparent discrepancy between this and the other results on the timing of localization of the CDC12 product is considered in the Discussion. It should be noted that the alpha factor experiments are subject to the caveat that alpha factor itself may perturb the normal distribution or assembly of the CDC3 or CDC12 protein (see also below).

Timing of Late Events at the Budding Site
We used three approaches to examine more closely the behavior of the CDC3 and CDC12 polypeptides at the end of the budding cycle. In the first approach, exponential-phase wild-type cells were examined by double-label immunofluorescence staining of tubulin and the CDC3 or CDC12 gene product. Cells were classified according to the appearance of their mitotic spindles, and the presence or absence of detectable rings of CDC3 or CDC12 antigen at the neck:was noted. Both proteins appeared to remain localized to the neck region so long as an intact spindle was maintained (Fig. 3, J and K). As the spindle began to break down (late nuclear division), the CDC12 protein seemed to disappear from the neck in most cells (Fig. 3, L and M). In contrast, the CDC3 protein seemed to be retained at the neck until very late stages, and probably until after nuclear division and cytokinesis were complete (Fig. 3, N and O). The results are summarized in Table II.. " In the second approach, we asked whether detectabletings of CDC3 and CDC12 pr0te~.ins~remained.~at the necks of/various temperature-sensiti~e cell dix, ision cycle mutants that.at= rest late in the budc~ing~ cyc!~ at restrictive: teml~rature (33). After 2-5 h at:restriofiv~ temperature, ~mutahtsarrested in medial.nuclear division (cdc/3, cdcl6, and cdc17) or in late nuclear division (cdc14 and cdc15) appeared consistently to maintain Structures that Contained both the CDC3 and CDC12 proteins (Fig. 5, A-F; and data not shown). Moreover, mutants arresting in very late nuclear division or between nuclear division and cytokinesis (cdc5 and cdc18) also appeared to maintain the localization of the CDC3 and CDC12 proteins (Fig. 5, CRY), although the results with cdc5 were somewhat heterogeneous. When strain 473D3 (cdc5-1) was grown at restrictive temperature for 2 h, by which time '~60% of the cells displayed large buds, detectable rings of CDC3 and CDC12 antigens were present in nea~ly all cells. Upon longer ~!ncubation at restrictive temperature, the population became very homogeneous (~95 % large-budded cells) as judged by phase-contrast microscopy, but the proportions of cells displaying ~detectable tings of CD.C3 or CDC12 antigen declined. By 5 h, only ,,o67 and ,y15% of the cells displayed detectable rings of the CDC3 and CDC12 antigens; respectively. Consideration of the detailed time courses in these experiments (not shown) indicated that most or all cells must have been arrested with large buds for >tl h before the antigen tings disappeared. In addition, cells of strain JPT176-H01 (cdc5-2) retained detectable rings of both antigens on essentially all cells even after 5 h at restrictive temperature (Fig. 5, I and M; and data not shown), although staining with CDC12-specific antibodies was weal Taken together, these observations suggest that disassembly of the CDC3-containing and CDC12-containing structures is not specifically programmed in cells that have reached the cdc5 arrest point, and that the gradual disappearance of these structures in strain 473D3 (and of the CDC12containing structures in strain JPT176-H01) reflects either a general cellular pathology or a slow leakage of the cells past the mutant block. The observation that tubulin immunofluorescence patterns also changed gradually in arrested 473D3 cellsAs consistent with either of the latter interpretations.   The third approach to investigation of events at the end of the budding cycle involved treating an exponentially growing population of MATa/MATa cells with alpha factor to prevent new budding (and thus, presumably, formation of new budding-associated rings of the CDC3 and CDC12 antigens), and following the disappearance of preexisting antigen rings as the cells completed division and arrested as unbudded cells. Although the results were somewhat variable (apparently due at least in part to variations in the effectiveness of the immunofluorescence staining), it was clear that the proportion of unbudded cells displaying rings of CDC3 antigen r~mained substantial (10---40%) throughout the 100-120 min in which new unbudded cells were being generated by cell division, and for '~20 min thereafter. Thus, the CDC3 protein appeared to linger in a ring structure at the budding site for 920 min after cell division under these conditions. The results obtained with the CDCl2-specific antibodies were similar, except that the proportions of unbudded cells displaying antigen rings were at all times smaller and more variable from experiment to experiment. Thus, the ring of CDCI2 polypeptide appeared to linger atthe budding site for only a short time after cell division.

Examination of CDC3 and CDC12 Proteins in Shmoos and Zygotes
The association of the CDC3 and CDC12 proteins with the sites of cell wall reorganization and chitin deposition in budcling cells suggested that these proteins might also be involved in the related processes that occur in cells that are mating or responding to mating pheromone (9, 26, 28, 36, 41, 42, 43; S. Ford and J. Pringle, manuscript in preparation). Indeed, after several hours of exposure to alpha factor, some shmoos (unbudded cells on which a broad protrusion has formed) exhibited faint but distinct bands of CDC3 and CDC12 immunottuorescence around the protruding regions (Fig. 5 O; and data not shown). This corresponded roughly with the regions of chitin deposition in these cells, as revealed by staining with Calcofluor (Fig. 5 P; reference 36). In mating cells, a faint band (or bands) of immunofluorescence in the region of fusion was often detectable soon after fusion with CDC3-specific antibodies (Fig. 5 Q, ceils a and b and regions of cells d and e noted by arrowheads). Again, this localization corresponded roughly with the region of chitin deposition (Fig. 5 R). These immunofluorescence patterns were not detectable in all shmoos and zygotes, and it was not always clear, that the structure stained was intracellular (see the problems with cell wall staining artifacts in re~rence 10). Thus, we cannot be certain that the association of thf CDC3 ar~ CDC12 proteins with the regions of ceil wall re~t',ganizatiqn is a normal feature of zygote and shmoo for-ma~t{on. It Was clear, however, that as zygotes prepared to bud they formed rings of CDC3 protein (Fig. 5 Q, ceils c and d) and CDCI2 protein (not shown) that persisted as the buds emerged and enlarged (Fig. 5 Q, cells e and f), essentially as seen in budding vegetative cells.

Discussion
To study the intracellular organization and function of the yeast CDC3 product, we raised antibodies against the ~sion proteins encoded by in-frame fusions of CDC3 to the E. coli lacZ and trpE genes. Antisera raised against each fusion pro-rein were able to recognize both fusion proteins; this both demonstrated the presence of CDC3-specific antibodies and /facilitated their effective affinity purification. Immunoblotring experiments using the purified antibodies revealed a single polypeptide of ,,o62 kD in extracts from normal yeast cells and a more abundant polypeptide of the same mobility in extracts from cells harboring a high-copy-number plasmid that contains the complete CDC3 coding region (11,25). The size of the presumed CDC3 product agrees well with the 60 kD predicted from the sequence (11). In addition, the antibodies revealed an abundant polypeptide of ,v50 kD in extracts from cells harboring a plasmid that appears to contain a CDC3 gone that is truncated at its 5' end (11). The size of this polypeptide is consistent with the hypothesis that its translation begins at the first available ATG (codon 70 of the normal CDC3 coding region; reference 11). The retention of cdc3-complemenfing activity by this plasmid (25) suggests that the amino-terminal segment of the CDC3 protein (a feature not shared with the other known members of this gene family; reference 11) is nonessential.
In view of the similarities in predicted amino acid sequence among the CDC3, CDCIO, CDC11, and CDC12 products (11), it is noteworthy that the CDC3-specific antibodies, like the CDCl2-specific antibodies (10), showed no apparent cross-reaction with the other three gene products. Althongh it is possible that further testing may reveal weak crossreactions, it seems likely that our immunofluorescence experiments have indeed revealed the localization of the CDC3 product and not of a weakly cross-reacting species. As in previous studies (10,23), affinity purification of the CDC3specific antibodies was essential to avoid misleading immunottuorescence results due to other antibodies, present in all rabbit sera tested, that were reactive with yeast cell constituents.
The immunottuorescence localization of the CDC3 product in wild-type cells revealed patterns similar to those observed with CDC/2-specific (10) and CDC/0-specific (H. B. Kim, S. Ketcham, and J. R. Pringle, manuscript in preparation) antibodies. These staining patterns suggest that all three gene products are associated with the ring of 10-nm filaments in the mother-bud neck. This hypothesis is supported by the immunofluorescence localization of the CDC]2 (10) and CDC3 products in mutant strains. Localization of the CDC3 product to the neck region was lost rapidly after a shift to restrictive temperature in cddO or cdcl2 mutants, but not in cddl or cdc4 mutants, consistent with electron microscopic observations on the behavior of the 10-nm illaments in these strains (1; Byers, B., and L. Goetsch. 1976.

J.
CellBiol. 70:35a; B. Byers and L. Goetsch, personal commmlication). Results with the cdc3 mutant were more difficult to interpret; staining of the neck region with CDC3specific antibodies was lost rapidly after the shift to restrictive temperature, although the electron microscopic observations suggest that the lO-nm filaments should remain in place until completion of the cell cycle in progress at the time of the temperature shift. Presumably, either the mutant CDC3 product remains in the filaments but becomes unreactive to the antibodies after the temperature shift, or it dissociates from the filaments without destroying their higher-order structure.
The most economical interpretation of the available data is that the CDC3, CDCIO, CDCI1, and CDC12 gene products are the primary constituents of the lO-nm filaments. As the predicted amino acid sequences of these polypeptides show no extensive similarities to those of other known filamentforming proteins (11), the yeast 10-urn filaments would thus represent a novel type of eukaryotic cytoskeletal element. However, it also remains possible that the CDC3, CDCIO, CDCll, and CDC12 products are all accessory proteins that associate with a filamentous structure composed primarily of some as yet unidentified, but more conventional, filamentforming protein.
Electron microscopic observations have suggested that the 10-nm filaments assemble at the budding site coincident with early bud emergence and disassemble rather abruptly just before cytokinesis (4,5). We attempted to explore further the assembly and disassembly processes using the CDC3specific and CDC12-specific antibodies. Interpretation of our results is subject to three major caveats. First, as noted above, we do not know the degree to which either protein is responsible for the higher-order structure of the filaments as observed in the electron microscope. Thus, the presence of the CDC3 and CDC12 products at a given site may not necessarily imply the presence of filaments at that site, and vice versa. Second, the electron microscopic studies themselves suffer from the uncertainty that the filaments can be difficult to see even in budded cells, and would have been very difficult to see in unbudded cells (B. Byers, personal communication). Thus, it is possible that the filaments are actually assembled before bud emergence, or remain assembled at the budding site after cytokinesis, at least in some cells. Third, apparent differences in the behavior of the CDC3 and CDC12 polypeptides might reflect instead differences in the affinities of the respective antibodies.
With these caveats in mind, the following comments can be made. First, immunofluorescence observations using wild-type, nuclear division-mutant,, and oL-factor-arrested cells consistently indicated that the CDC3 product remains localized to a ring structure at the old budding site on both mother and daughter cells after cytokinesis. The corresponding observations with respect to the CDC12 product were less consistent (perhaps reflecting the variable efficacy of the immunofluorescence staining), but in aggregate suggest that this polypeptide may behave similarly. It appears that the CDC3 and CDC12 antigen rings then disappear gradually during the subsequent unbudded phase, at rates that may differ for CDC3 and CDC12, and that may also vary from ceil to cell. If we take the electron microscopic observations at face value, our results thus suggest that the higherorder structure of the filaments may disassemble before cytokinesis even though at least some of the filamentassociated polypeptides remain localized to the region where the filaments had been. If there is a specific point in the cell cycle at which filament disassembly is triggered, it is apparently not reached in any of the mutants blocked in medial or late stages of nuclear division, as electron microscopic (Byers, B., and L. Goetsch. 1976. J. Cell BioL 70:35a; B. Byers, personal communication) and immunofluorescence observations are consistent in indicating that filaments and filament-associated polypeptides remain in place at the mother-bud neck in arrested cells of these mutants. Cells blocked in nuclear division by the microtubule-disassembling drug nocodazole also retain 10-nm filaments at the motherbud neck for extended periods, as judged by EM (19).
Second, immunofluoreseence observations on exponen-tially growing and synchronized cultures consistently indicated that the CDC3 product is localized to a ring structure at the presumptive budding site well before chitin-ring formarion and the formation of buds visible by light microscopy (1>15 rain under the growth conditions used). The corresponding observations for CDC12 were again less consistent, but in aggregate suggest that this polypeptide also may be localized to the budding site at least a few minutes before bud emergence. If we take the electron microscopic observations at face value (but note the caveat above), our results thus suggest that the higher-order structure of the filaments may assembly only some time after the known filament-associated polypeptides have been localized to the budding site.
In any case, the immunofluorescence observations appear to clarify the possible role of the 10-nm filaments in formation of the chitin ring. Such a role had been suggested by the evidence that the cdc3, cdclO, cdcll, and cdc12 mutants, which lack the 10-nm filaments, also fail to form normal chitin rings, whereas other cdc mutants such as cdc4, which possess apparently normal 10-nm filaments, also form normal-looking chitin rings (Fig. 3, C, E, G, and H; Byers, B., and L. Goetsch. 1976. J. Cell Biol. 70:35a; references l, 10; B. Byers, personal communication). This correlation might mean either that chitin-ring formation is dependent on the 10-urn filaments or the reverse, but support for the former interpretation was provided by the observations (a) that the CDC3, CDCIO, and CDCI2 gene products all localize to the vicinity of the 10-nm filaments; and (b) that the cdclO and cdc12 mutants lose preexisting filaments after a shift to restrictive temperature; taken together, these data suggest strongly that the primary lesions in these mutants involve formation of the 10-nm filaments. Moreover, a chsl::URA3 csd2-1 mutant strain (3; C. Bulawa, personal communication), which produces little or no chitin, nonetheless shows apparently normal localization of the CDC3, CDCIO, and CDC12 products (our unpublished results), suggesting that filament formation is not dependent on the presence of a chitin ring. However the putative role for the 10-nm filaments in formation of the chitin ring was difficult to reconcile with the electron microscopic evidence that the filaments only form as the bud is emerging (5), whereas the chitin ring is well developed by that time (Fig. 4 A; references 16,21). From our immunofluorescence studies, it now seems clear that at least some of the filament-associated polypeptides, if not actually the filaments themselves, are assembled at the budding site in time to play a role in chitin ring formation. However, it should also be noted that the 10-urn filaments and their associated polypeptides cannot be the only factors determining where chitin is deposited, as these elements appear equilaterally on both sides of the neck, whereas chitin is deposited only in the wall of the mother cell. A further consideration is the formation of septum-like structures at aberrant locations in cdc3, cddO, cdc11, and cdc12 mutants (38); one interpretation of these observations is that the 10-nm filaments are necessary for the normal positioning of the chitin ring, but not actually for its formation.
An approximate correlation between the distribution of the CDC3 and CDC12 products and the sites of cell wall reorganization and chitin deposition was also observed (with some significant uncertainties; see Results) in shmoos and zygotes. It is not clear whether the apparently diffuse localization of the CDC3 and CDC12 polypeptides in these ceils actually reflects the formation of 10-nm filaments in the corresponding regions, or whether these polypeptides can function without forming filaments.
The observation that the CDC3 polypeptide, at least, appears at the presumptive budding site well in advance of bud emergence raises the very interesting questions of whether it is the 10-urn-filament-associated polypeptides or the actin network (2, 21, 30) that assembles first at the budding site, and of whether the later of these events is dependent upon the earlier. It should be noted that reorganizations of the actin network and of the 10-nm-filament-associated polypeptides are also temporally correlated at the end of the budding cycle (see above and refs. 2, 21, 30) and in shmoos and zygotes (see above; ref. 15; and S. Ford and J. Pringle, manuscript in preparation). We are currently investigating these issues.