Ploidy Cells Research Articles

Hyperthermo-radiosensitivity of esophageal cancer cells with high DNA ploidy in vitro.

The in vitro sensitivity to hyperthermia and radiation of esophageal cancer cells obtained from 50 patients was assayed by using the in vitro succinate dehydrogenase inhibition test, and the findings were correlated with data on DNA analysis. The DNA distribution patterns were grouped into types I, II, III and IV, according to the frequency of aneuploid cell populations. Esophageal cancer cells of high ploidy (type IV) had a lower sensitivity to radiation, however, a greater sensitivity to hyperthermia as compared to cells of low ploidy (type II). The sensitivity to hyperthermia was determined as positive in 2 of 8 for type II, in 7 of 25 for type III, and in 9 of 17 for type IV. In contrast, the positive sensitivity rates to radiation in type II, III, and IV were 25.0, 8.0, and 5.9%, respectively. When cells were exposed to combination of radiation with hyperthermia, the positive sensitivity rates increased in all groups (50.0, 44.0 and 70.6% in type II, III, and IV, respectively). There was a significant correlation between mean DNA values and the SD activities following exposure to heat treatment or radiation. These data, describing that cancer cells of high ploidy might have greater sensitivity to hyperthermia compared to radiation, indicate the clinical benefits of hyperthermia in cases of esophageal carcinoma, especially for patients with high DNA ploidy.

Placental site trophoblastic tumor: Immunohistochemical and nuclear DNA study

A rare case of placentae site trophoblastic tumor (PSTT) studied by immunohistochemistry and nuclear DNA analysis is reported. The patient, a 24-year-old Japanese female, complained of amenorrhea. Dilatation and curettage revealed a small specimen that contained trophoblastic cells and caused intractable bleeding. Pelvic sonography revealed a 5-cm mass in the posterior uterine wall with multiple cystic lesions of several sizes. The cystic lesions were shown to be dilated vessels by magnetic resonance imaging (MRI) and digital subtraction angiography (DSA). Serum β-hCG (β subunit of human chorionic gonadotropin) was 3.7 ng/ml. Total abdominal hysterectomy revealed a well-circumscribed, yellow, soft mass in the posterior uterine wall. Microscopic findings were consistent with PSTT and the mitotic count was extremely low. Immunohistochemically, most of the tumor cells were intensely stained with human placental lactogen, whereas few were stained with human chorionic gonadotropin. The nuclear DNA content of the trophoblastic cells showed a sharp peak at the triploid range coexistent with a few cells of higher ploidy. This is the first report of sonographic findings and nuclear DNA analysis by spot cytometry in a case of PSTT.

Analysis of megakaryocyte ploidy in patients with thrombocytosis

The DNA content of bone marrow megakaryocytes was analyzed in 24 patients with myeloproliferative disorders, 23 patients with secondary thrombocytosis and 15 normal volunteers using 2-color flow cytometry. Compared with normal controls, the majority of patients with secondary thrombocytosis, polycythemia vera and essential thrombocytosis exhibited a relative increase in higher ploidy (greater than 16N) cells. In contrast, patients with chronic myelogenous leukemia exhibited an increase in lower ploidy cells (less than 16N), with a modal DNA content of 8N. Patients with myeloproliferative disorders tended to show a decrease in the 16N megakaryocyte population compared with patients with secondary thrombocytosis. No correlation between ploidy distribution and platelet count was observed.

Flow Cytometric Analysis of Megakaryocytes From Patients With Abnormal Platelet Counts

Megakaryocytes (MKs) from 40 patients with quantitative platelet disorders and 19 normal volunteers were analyzed by flow cytometry for size, fine cell internal structure and granularity, membrane expression of the glycoprotein (GP) IIb/IIIa complex, and for ploidy distribution. Analysis was performed on unfractionated minimally manipulated marrows obtained from routine bone marrow aspirates. MKs were labeled with a fluorescent lineage-specific monoclonal antibody to the GPIIb/IIIa complex followed by DNA staining with propidium iodide. Eight hundred to 3,000 MKs were analyzed in each sample. The modal ploidy distribution in normals was 16N, comprising about half of the megakaryocytic population, with 22.6% of the cells less than or equal to 8N and 22.0% greater than or equal to 32N. Twelve thrombocytopenic patients with decreased marrow MKs on biopsy (mean platelet count [MPC] 44,600/microliters) showed an increase in low ploidy cells with 53.2% less than or equal to 8N (P less than .01); cell size was reduced in three patients when compared to normal cells of identical ploidy (P less than .05). Eight thrombocytopenic patients with enhanced platelet destruction (with normal or increased MKs on biopsy and shortened platelet survival; MPC 41,400/microliters) showed an increased proportion of high ploidy cells greater than or equal to 32N to 39.2% (P less than .01). Increased cell size and granularity were found in four of these patients (P less than .05). Six patients with thrombocytopenia secondary to multiple mechanisms affecting both platelet production and destruction (MPC 66,700/microliters) showed no shift in ploidy. Four patients with primary thrombocytosis (two with thrombocythemia and two with polycythemia vera; MPC 822,500/microliters) showed a marked shift toward high ploidy cells with 42.3% greater than or equal to 32N and 7.6% greater than or equal to 64N cells (P less than .01). The shift was accompanied by a marked increase in cell size and granularity in the patients with thrombocythemia. Ten patients with thrombocytosis secondary to chronic blood loss, malignant or inflammatory disorders (MPC 714,000/microliters), showed variable distributions with four patients exhibiting a shift in ploidy to the right similar to that found in the patients with increased platelet destruction. Based upon the present data, flow cytometric ploidy distribution may be diagnostically useful in thrombocytopenic patients by discriminating between disorders of platelet production and destruction. (ABSTRACT TRUNCATED AT 400 WORDS)

Flow cytometric analysis of megakaryocytes from patients with abnormal platelet counts

Megakaryocytes (MKs) from 40 patients with quantitative platelet disorders and 19 normal volunteers were analyzed by flow cytometry for size, fine cell internal structure and granularity, membrane expression of the glycoprotein (GP) IIb/IIIa complex, and for ploidy distribution. Analysis was performed on unfractionated minimally manipulated marrows obtained from routine bone marrow aspirates. MKs were labeled with a fluorescent lineage-specific monoclonal antibody to the GPIIb/IIIa complex followed by DNA staining with propidium iodide. Eight hundred to 3,000 MKs were analyzed in each sample. The modal ploidy distribution in normals was 16N, comprising about half of the megakaryocytic population, with 22.6% of the cells less than or equal to 8N and 22.0% greater than or equal to 32N. Twelve thrombocytopenic patients with decreased marrow MKs on biopsy (mean platelet count [MPC] 44,600/microliters) showed an increase in low ploidy cells with 53.2% less than or equal to 8N (P less than .01); cell size was reduced in three patients when compared to normal cells of identical ploidy (P less than .05). Eight thrombocytopenic patients with enhanced platelet destruction (with normal or increased MKs on biopsy and shortened platelet survival; MPC 41,400/microliters) showed an increased proportion of high ploidy cells greater than or equal to 32N to 39.2% (P less than .01). Increased cell size and granularity were found in four of these patients (P less than .05). Six patients with thrombocytopenia secondary to multiple mechanisms affecting both platelet production and destruction (MPC 66,700/microliters) showed no shift in ploidy. Four patients with primary thrombocytosis (two with thrombocythemia and two with polycythemia vera; MPC 822,500/microliters) showed a marked shift toward high ploidy cells with 42.3% greater than or equal to 32N and 7.6% greater than or equal to 64N cells (P less than .01). The shift was accompanied by a marked increase in cell size and granularity in the patients with thrombocythemia. Ten patients with thrombocytosis secondary to chronic blood loss, malignant or inflammatory disorders (MPC 714,000/microliters), showed variable distributions with four patients exhibiting a shift in ploidy to the right similar to that found in the patients with increased platelet destruction. Based upon the present data, flow cytometric ploidy distribution may be diagnostically useful in thrombocytopenic patients by discriminating between disorders of platelet production and destruction.

Ultrastructural changes in nucleoli and fibrillar centers under the effect of local ultraviolet microbeam irradiation of interphase culture cells

As shown previously, ultraviolet (uv) microbeam irradiation of one of the two mature nucleoli within an interphase cell nucleus causes significant diminution and inactivation of the irradiated nucleolus and compensatory growth and activation of the nonirradiated one. In the present work we describe the results of an ultrastructural study of this phenomenon. The changes in the nucleoli were examined by means of complete series of ultrathin sections obtained from seven irradiated pig kidney cells. The compensatory hypertrophy of the nonirradiated nucleoli is shown to be accompanied by a nearly twofold increase in the number of fibrillar centers (FCs) and by a decrease in their linear dimensions compared with the control cells of the same ploidy. In the degraded nucleoli the number of FCs decreases, but their dimensions increase. Ultraviolet microbeam irradiation causes dramatic diminution of the dense fibrillar component within the irradiated nucleoli as well. The nucleolar capacity for compensatory hypertrophy indicates that in addition to active ribosomal genes, mature nucleoli also contain “silent” genes capable of being activated under extreme conditions to sustain the required level of rRNA synthesis. It is assumed that activation of latent ribosomal genes is accompanied by FC “fragmentation” without a considerable increase in their total volume per cell.

Endpolyploidy and digestion in the midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae).

The Feulgen-DNA content of the midgut cell nuclei of Rhodnius prolixus is estimated by microspectrophotometry. Midgut cells contain 23.35-32.16 pg DNA/nucleus, which represents an increase of up to 26c when compared to diploid R. prolixus brain tissue. A high copy number cell population disappears from the anterior intestinal cells after feeding, and the presence of a new population of low copy number cells indicates that there may be some regeneration of the midgut epithelium in adult insects, or that non-mitotic division of the high ploidy cells occurs. Polyploidy of the midgut cells is thought to be important in the rapid post-feeding synthesis of digestive proteinases, whereby enzymes are produced without the immediate need for DNA synthesis.

Comparative enzyme and ethanol production in an isogenic yeast ploidy series

Effects on ethanol production by increases in gene dosage independent of heterosis in yeast are compared for an isogenic ploidy series ranging from haploid to tetraploid. The per-cell rate of ethanol accumulation in parallel batch cultures increases with cell ploidy, and is attributable to intrinsic, ploidy-associated increases in cell mass-adjusted ethanol production rates. This increase in per-cell ethanol accumulation in the tetraploid strain is as high as 6.9 times the level of accumulation in the haploid. That is, the efficiency of ethanol production per unit cell mass is greater in cells of higher ploidy.

Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration

In an attempt to achieve accurate quantification of DNA levels in cell nuclei, we studied the influence of salt concentration on the fluorescence of cell nuclei complexed with Hoechst-33258 (Hoe) fluorochrome. The fluorescence of cell nuclei was compared with that of extracted DNA as well as that of nucleosome core. Conformational changes in these complexes were examined by measuring both fluorescence anisotropy and fluorescence lifetime in the nanosecond region. The results showed that the fluorescence of DNA-Hoe was quenched by the nucleosomal structure, there being an associated increase in anisotropy and a decrease in the fluorescence lifetime; however, the fluorescence was restored to the original level by the addition of a high concentration of NaCl, CsCl, or LiCl. The reduction in fluorescence may have been due to loss of fluorescence energy caused by collision of the fluorophore with histones in the nucleosome. The addition of 1 M NaCl to the medium used for staining with Hoe greatly stabilized the fluorescence of DNA in cell nuclei. The DNA content of individual cell nuclei was determined by comparing the fluorescence of these nuclei with that of a standard DNA solution. For lymphocytes and liver ploidy cells, reasonably accurate values were obtained by applying the present method.

Changes in ploidy level of epidermal cells during last larval instar of the tobacco hornworm, Manduca sexta.

The relative DNA content of Manduca sexta abdominal epidermal nuclei during the final larval instar was measured by cytophotometry of whole-mount preparations of the epidermis. In the middle intrasegmental region, epidermal cells showed a ploidy level of 4C to 32C on the day of ecdysis. During the subsequent period of feeding, the proportion of higher ploidy cells, such as 16C and 32C, increased. This situation remained until the day of apolysis preceding pupal cuticle formation when mitoses reduced the cells to 2C, 4C, 8C and 16C, except for the pupal pock-mark cells, which increased to 32C or 64C. Metaphase cells showed various ploidy levels, correlated with the size of their mitotic figures. By contrast, in the anterior and posterior margin of a segment where no mitoses occurred, the cells continued to increase in ploidy throughout the instar.

Separation of murine megakaryocytes and their progenitors on continuous gradients of Percoll.

Murine bone marrow was separated on continuous Percoll density gradients to analyze the distribution of cells of the megakaryocyte lineage. Eighty-seven percent of the recovered megakaryocytes were found in fractions of density less than 1.058 g/cm3, with 63% of these cells found between 1.020 and 1.036 g/cm3. When megakaryocytes were classified according to size, 92% of the large (greater than or equal to 18 micron) acetylcholinesterase (AchE) positive cells were found in the least dense fractions (1.016-1.039 g/cm3), whereas 86% of the small (less than or equal to 10.6 micron) AchE positive cells were found in fractions of higher density (1.039-1.078 g/cm3). The distribution of enzymatic AchE activity of the separated fractions corresponded to the location of the histochemically positive cells. When ploidy measurements were made of various fractions, most of the high ploidy (32N and 64N) cells were found at low density (1.028-1.036 g/cm3), whereas no cells greater than 4N were found at density greater than 1.071 g/cm3. Thus, large AchE positive cells and the cells of highest ploidy were found at lower densities of Percoll, while small AchE positive cells and cells of low ploidy were found at higher densities. An exception to this inverse relationship was found in fractions of lowest density (less than 1.030 g/cm3) where an anomalous distribution of size and ploidy was found. The majority of megakaryocytic colony-forming cells (CFU-MK) were found at high density, as were the granulocyte-macrophage colony-forming cells (CFU-GM; approximately 1.074 g/cm3). The density distribution of the incorporation of tritiated thymidine into liquid marrow cultures was concordant with the high density distribution of colony-forming cells. The data show that megakaryocytic maturity and Percoll density varies inversely and that fractionation of marrow on continuous Percoll gradients may be a useful method for the separation and/or enrichment of megakaryocytes at different stages of differentiation.

Uptake and disposition of mirex in hepatocytes and subcellular fractions in CD1 mouse liver.

In vivo uptake and disposition of [14C]mirex by CD1 mouse liver subcellular fractions and cells of different nuclear ploidy were examined following single or multiple doses of mirex injected intraperitoneally. Significant amounts of mirex were rapidly taken up by liver (21-29%), suggesting that liver is one of the primary sites of accumulation of the chemical. Among subcellular fractions, mirex was predominantly distributed in mitochondria and microsomes in the irreversibly bound form (about 20%), although its levels fluctuated considerably with time. Mirex was completely dissociated with trichloroacetic acid treatment from both nuclear and plasma membrane fractions, although the total uptake by these fractions was markedly high. The time course of uptake and concentration-dependent disposition of mirex revealed that polyploid hepatocytes selectively accumulated higher amounts of the chemical (two to three times) compared to diploid hepatocytes. The increased affinity of polyploid cells to mirex may indicate a greater susceptibility of this cell type to the chemical insult and also may suggest a possible early involvement of polyploids in the tumorigenic process in rodent livers.

The influence of juvenile hormone on polyploidy and vitellogenesis in the fat body of Locusta migratoria.

Abstract Somatic polyploidy is a common developmental feature of endocrine target tissues, including vertebrate liver and insect fat body. Vitellogenin (Vg) synthesis in locust fat body is under strict control of juvenile hormone (JH) and is correlated with DNA synthesis and massive polyploidization of the tissue. We have used this system to concurrently monitor interactions between hormone, specific protein synthesis, and generation of cells with defined ploidy levels. Normal maturation of both males and females were compared, as were animals treated with precocene III and the JH analog ZR-515. Following are the main results and observations: Polyploidization occurs in both males and females but is much more pronounced in the latter. Peak DNA synthesis occurs in females just before onset of Vg synthesis and is coincident with predominant appearance of octoploid (8c) cells. In males DNA synthesis peaks earlier than in females, during transition from 2 c to 4 c cells, and then declines. Suppression of JH production by precocene prevents Vg synthesis completely and also inhibits DNA synthesis and generation of higher ploidy cells in both sexes. Reexposure of withdrawn animals to hormone reversed the foregoing conditions, but the disparity between sexes in timing of DNA synthesis and bulk tissue ploidy level was maintained. The developmental significance of these results is discussed.

Kinetics of cell proliferation in a rapidly growing murine lymphosarcoma.

The growth kinetics of a murine lymphosarcoma were investigated the spleen and liver of mice by means of quantitative flow-cytofluorometric DNA measurements in individual cells in combination with 3H-thymidine incorporation into the DNA. The amount of DNA in the S-phase cells was calculated from the resulting DNA histograms. From the 3H-thymidine incorporation, related to the amount of S-phase DNA, the relative changes in the duration of the S-phase during the growth of the lymphosarcoma were estimated. A marked prolongation of the duration of the S-phase was observed in the course of the growth of the lymphosarcoma. The disappearance of cells of higher ploidy normally found in the liver indicates the elimination of those normal cells in the course of the tumour growth.

Excessive reversible phenobarbital induced nuclear DNA-polyploidization in the growing mouse liver.

Phenobarbital was injected intraperitoneally into male white NMRI mice aged 0.5, 1. 1.5, 3, 6 and 12 months at a dose of 120 mg/kg body weight for 10 consecutive days. The 0.5 month-old mice did not tolerate the phenobarbital dose and died. The experimental animals and one of the controls were sacrificed 1, 3, 5, 10, 15 and 20 days after phenobarbital administration was started. Liver weights were recorded and liver cells were isolated. The number of nuclei per cell was determined and the DNA-content of each single nucleus was measured by Feulgen fluorescence cytophotometry. Liver weights showed an increase of 25--30% during phenobarbital treatment and returned slowly to lower values after cessation of drug application. The hepatic enlargement was accompanied by an excessive and likewise reversible nuclear and whole cell DNA-polyploidization, i.e. polyploidization beyond the physiological age-dependent ploidy level; for example, mean values of 7.7 c per nucleus (versus 4.2 c in the controls) and 14.3 c for whole liver cells (versus 7.5 c in the controls) were found in 3 months-old animals after 5 days of treatment. As with the induction of microsomal enzymes, the augmentation of smooth endoplasmic reticulum, and the increase of RNA- and protein-synthesis, excessive DNA-polyploidization of liver cell nuclei appears to be an expression of hepatocellular hypertrophy due to the functional or metabolic stress imposed upon the liver by large quantities of phenobarbital. After cessation of drug administration the abnormally high ploidy cells are eliminated - probably by necrobiosis - and the liver cells return to their normal age-dependent DNA-ploidy level. The liver cells of the one-month-old animals revealed the physiological polyploidization to be slightly inhibited. This is probably due to some toxic effect of phenobarbital. Phenobarbital did not alter the number of nuclei per liver cell.

Flow Cytometric Analysis of Megakaryocyte Ploidy. Comparison With Feulgen Microdensitometry and Discovery That 8N is the Predominant Ploidy Class in Guinea Pig and Monkey Marrow

Flow cytometry (FCM) was adapted to measure ploidy values in megakaryocytes isolated from normal guinea pigs and African green monkeys. The FCM approach was validated by microscopic identification of electronically sorted 8N and higher ploidy cells and by comparisons with Feulgen microdensitometry on the same samples. Sorted 8N populations contained only megakaryocytes and were completely free of cell clumps; clumping was also absent in cell suspensions examined prior to FCM. Discrete megakaryocyte peaks were found at 8N, 16N, and 32N but not at intermediate values. The ploidy patterns of isolated megakaryocytes were similar by both methods (approximately 15% 8N, 58% 16N, and 27% 32N in guinea pigs). Unexpectedly, FCM analysis of unseparated marrow cell suspensions found that the most frequent ploidy class of megakaryocytes was 8N (50%). In contrast, by microdensitometry, the 8N class accounted for only 21% of the megakaryocytes in unseparated marrow cell suspensions. Since the traditional method depends on measurement only of easily recognizable megakaryocyte nuclei, a microdensitometric analysis of unselected marrow cells was carried out. Many more smaller 8N megakaryocytes (48%) were found by this approach, confirming the FCM data and the bias of the traditional sampling method. The decrease in 8N megakaryocytes (50%–15%) with enrichment was accounted for by the consistent finding that cells lost during isolation were predominantly 8N. with relative sparing of the 32N class. FCM provides more rapid and objective analysis of ploidy patterns in much larger samples of megakaryocytes than previous methods.

Flow cytometric analysis of megakaryocyte ploidy. Comparison with Feulgen microdensitometry and discovery that 8N is the predominant ploidy class in guinea pig and monkey marrow

Flow cytometry (FCM) was adapted to measure ploidy values in megakaryocytes isolated from normal guinea pigs and African green monkeys. The FCM approach was validated by microscopic identification of electronically sorted 8N and higher ploidy cells and by comparisons with Feulgen microdensitometry on the same samples. Sorted 8N populations contained only megakaryocytes and were completely free of cell clumps; clumping was also absent in cell suspensions examined prior to FCM. Discrete megakaryocyte peaks were found at 8N, 16N, and 32N but not at intermediate values. The ploidy patterns of isolated megakaryocytes were similar by both methods (approximately 15% 8N, 58% 16N, and 27% 32N in guinea pigs). Unexpectedly, FCM analysis of unseparated marrow cell suspensions found that the most frequent ploidy class of megakaryocytes was 8N (50%). In contrast, by microdensitometry, the 8N class accounted for only 21% of the megakaryocytes in unseparated marrow cell suspensions. Since the traditional method depends on measurement only of easily recognizable megakaryocyte nuclei, a microdensitometric analysis of unselected marrow cells was carried out. Many more smaller 8N megakaryocytes (48%) were found by this approach, confirming the FCM data and the bias of the traditional sampling method. The decrease in 8N megakaryocytes (50%–15%) with enrichment was accounted for by the consistent finding that cells lost during isolation were predominantly 8N. with relative sparing of the 32N class. FCM provides more rapid and objective analysis of ploidy patterns in much larger samples of megakaryocytes than previous methods.

Distribution of membrane-bound and free ribosomes in growing yeast

1. The distribution of membrane-bound and free ribosomes was investigated in stationary as well as in growing yeast cells. The relative amount of free ribosomes varies with the growth phase of the cell culture. During the duplication phases of the cell, relative maxima of free ribosomes can be found. However, the absolute amount of free ribosomes is fairly constant during the growth of the cells. 2. Membrane-bound ribosomes show lower polypeptide synthesis activity in a cell-free, poly(U)-dependent system than free ribosomes. 3. There is no difference in the distribution pattern of free and membrane-bound ribosomes in growing yeast cells of different ploidy. 4. A turnover between free and membrane-bound ribosomes is suggested to be in agreement with the hypothesis of Branes and Pogo ((1975) Eur. J. Biochem. 54, 317–328).

Chapter 3 Methods in Sporulation and Germination of Yeasts

Publisher Summary This chapter describes methods in sporulation and germination of yeasts. Sporulation of yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe provides an attractive system for the study of unicellular differentiation, especially the processes of meiosis and genetic recombination. Yeasts are easily cultured, sporulated, and germinated and are quite amenable to genetic analysis. Sporulation occurs when cells preadapted to oxidative growth are incubated in a medium such as 1% potassium acetate in the absence of a nitrogen source. Sporulation occurs in diploids or cells of higher ploidy that carry both a- and α-mating type alleles, diploids of genotype a/a or α/α do not sporulate. Sporulation is distinctly different from the vegetative cell cycle, as the conditions that support vegetative growth do not support sporulation of strains of S. cerevisiae. Sporulation occurs under pseudostarvation conditions in which the cells do not proliferate. During sporulation, cells undergo meiosis rather than mitosis and yield four haploid genetic complements enclosed in four refractile spores within an ascus. The chapter identifies areas toward which major attention has been directed in study of sporulation and germination. Identification of stages during sporulation is described and isolation of mutants specific for sporulation is explained. Methods for measuring germination and outgrowth are also described in the chapter.

Chapter 18 Mutants of Meiosis and Ascospore Formation

Publisher Summary This chapter focuses on mutants of meiosis and ascopore formation. Formation of spores by both prokaryotic and eukaryotic microbes presents an opportunity to study the biochemical and genetic regulation of differentiation in single cells. Sporulation of yeasts is of particular interest, because it encompases meiosis and ascospore development. A comprehensive investigation of the genetic control of sporulation requires procedures that permit the detection of both recessive and dominant gene mutations. The detection of recessive mutations affecting meiosis and ascospore formation is complicated by the fact that these processes occur in diploid cells or cells of higher ploidy. Two systems have been developed that allow the recovery of both recessive and dominant mutations. One approach takes advantage of the life cycle of homothallic strains to obtain mutants in the homozygous condition in diploid cells. This procedure has been used with both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Another technique employs certain n + 1 disomic strains of S. cerevisiae that begin but do not complete sporulation. In this chapter, detailed methods of isolation and characterization of mutants with defective sporulation are presented. The properties of the mutants and the information they have yielded regarding the control of sporulation functions are also discussed.