Nuclear migration in Saccharomyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein.

We have isolated a novel gene (NUM1) with unusual internal periodicity. The NUM1 gene encodes a 313 kDa protein with a potential Ca2+ binding site and a central domain containing 12 almost identical tandem repeats of a 64 amino acid polypeptide. num1-disrupted strains grow normally, but contain many budded cells with two nuclei in the mother cell instead of a single nucleus at the bud neck, while all unbudded cells are uninucleate. This indicates that most G2 nuclei divide in the mother before migrating to the neck, followed by the migration of one of the two daughter nuclei into the bud. Furthermore, haploid num1 strains tend to diploidize during mitosis, and homozygous num1 diploid or tetraploid cells sporulate to form many budded asci with up to eight haploid or diploid spores, respectively, indicating that meiosis starts before nuclear redistribution and cytokinesis. Our data suggest that the NUM1 protein is involved in the interaction of the G2 nucleus with the bud neck.     

Yeast Num1p associates with the mother cell cortex during S/G2 phase and affects microtubular functions

The NUM1 gene is involved in the control of nuclear migration in Saccharomyces cerevisiae. The content of NUM1 mRNA fluctuates during the cell cycle, reaching a maximum at S/G2 phase, and the translation product Num1p associates with the cortex of mother cells mainly during S, G2, and mitosis, as seen by indirect immunofluorescence. The nuclear spindle in NUM1-deficient large-budded cells often fails to align along the mother/bud axis, while abnormally elongated astral microtubules emanate from both spindle pole bodies. A num1 null mutation confers temperature sensitivity to the cold-sensitive alpha-tubulin mutant tub1- 1, and shows synthetic lethality with the beta-tubulin mutant alleles tub2-402, tub2-403, tub2-404, and tub2-405. Deletion mapping has defined three functionally important Num1p regions: a potential EF hand Ca2+ binding site, a cluster of potential phosphorylation sites and a pleckstrin homology domain. The latter domain appears to be involved in targeting Num1p to the mother cell cortex. Our data suggest that the periodically expressed NUM1 gene product controls nuclear migration by affecting astral microtubule functions.     

Physiological analysis of mutants indicates involvement of the Saccharomyces cerevisiae GPI-anchored protein gp115 in morphogenesis and cell separation.

This paper reports a phenotypic characterization of ggp1 mutants. The cloned GGP1 (GAS1) gene, which encodes a major GPI-anchored glycoprotein (gp115) of Saccharomyces cerevisiae of unknown function, was used to direct the inactivation of the chromosomal gene in haploid and diploid strains by gene replacement. The analysis of the null mutants reveals a reduction in the growth rate of 15 to 40%. Cells are round, with more than one bud, and extensively vacuolized. In the stationary phase, mutant cells are very large, arrest with a high percentage of budded cells (about 54 and 70% for haploid and diploid null mutants, respectively, in comparison with about 10 to 13% for control cells), and have reduced viability. The observed phenotype suggests defects in cell separation. Flow cytometric analysis of DNA reveals an increase in the fraction of cells in the G2+M+G1* compartment during exponential growth. Conjugation and sporulation are not affected. The exocellular location of gp115 led us to examine cell wall properties. Cell wall and septum ultrastructure of abnormally budded cells was analyzed by electron microscopy analysis, and no appreciable differences from wild-type cells were found. Microscopic analysis revealed an increase in chitin content and delocalization. In comparison with control cells, ggp1 null mutants are shown to be resistant to Zymolyase during the exponential growth phase. A fivefold overexpression of gp115 does not bring about any effects on cell growth parameters and cell wall properties.     

Mating-Type Functions for Meiosis and Sporulation in Yeast Act through Cytoplasm

Given a nutritional regime marked by a low nitrogen level and the absence of fermentable carbon sources, conventional a/α diploid cells of Saccharomyces cerevisiae exhibit a complex developmental sequence that includes a round of premeiotic DNA replication, commitment to meiosis and the elaboration of mature tetrads containing viable ascospores. Ordinarily, haploid cells and diploid cells of genotype a/a and α/α fail to display these reactions under comparable conditions. Here, we describe a simple technique for sporulation of α/α and a/a cells. Cells of genotype α/α are mated to haploid a cells carrying the kar1 (karyogamy defective) mutation to yield heterokaryons containing the corresponding diploid and haploid nuclei. The kar1 strains mate normally, but nuclei in the resultant zygotes do not fuse. When heterokaryotic cells are inoculated into sporulation media, they produce asci with six spores. Four spores carry genotypes derived from the diploid nucleus and the other two possess the markers originating from the haploid nucleus, i.e., the diploid nucleus divides meiotically while the haploid nucleus apparently divides mitotically. Similarly, the a/a genome is "helped" to sporulate as a consequence of mating with α kar1 strains. The results allow us to conclude that the mating-type functions essential for meiosis and sporulation are communicated and act through the cytoplasm and that sporulation can be dissociated from typical meiosis. This procedure will facilitate the genetic analysis of strains that are otherwise unable to sporulate.     

Mal3, the Schizosaccharomyces pombe homolog of EB1, is required for karyogamy and for promoting oscillatory nuclear movement during meiosis

Two successive rounds of chromosome segregation following a single round of DNA replication enable the production of haploid gametes during meiosis. In the fission yeast Schizosaccharomyces pombe, karyogamy is the process where the nuclei from 2 haploid cells fuse to create a diploid nucleus, which then undergoes meiosis to produce 4 haploid spores. By screening a collection of S. pombe deletion strains, we found that the deletion of 2 genes, mal3 and mto1, leads to the production of asci containing up to 8 spores. Here, we show that Mal3, the fission yeast member of the EB1 family of conserved microtubule plus-end tracking proteins, is required for karyogamy, oscillatory nuclear movement, and proper segregation of chromosomes during meiosis. In the absence of Mal3, meiosis frequently initiates before the completion of karyogamy, thus producing up to 8 nuclei in a single ascus. Our results provide new evidence that fission yeast can initiate meiosis prior to completing karyogamy.     

Cell morphology, nucleus distribution and DNA content in haploid and diploid Aspergillus niger strains

The cells of haploid Aspergillus niger strains contain, on the average, 7-9 nuclei, a fragment of a thin hypha 100 me long comprising 11-19 nuclei. The cells of a diploid strain are 1.5-2.6 times larger in volume. The diploid cells contain less nuclei and more cytoplasm per nucleus as compared to the haploid strains. The primary sterigmae of Aspergillus niger comprise 3-13 nuclei, the secondary sterigmae and conidia, one nucleus. The conidia of the diploid strains are 1.8-2.0 times larger in volume and contain twice as much DNA as compared to the haploid strains.     

The interrelationship of cell growth and division in haploid and diploid cells of Saccharomyces cerevisiae

The coordination of cell growth and division has been examined in isogenic haploid and diploid strains of Saccharomyces cerevisiae. The average cell volume of the haploid and diploid cells was unaffected by a range of environmental conditions and generation times. For most environments and generation times the mean cell volume of diploid cells was between 1.52 and 1.83 of the haploid cell volume. Both haploid and diploid cell volumes were reduced drastically when the cells were grown in the chemostat with glucose as the limiting substrate. In this environment diploid cells have the same mean cell volume as haploid cells. Diploid cells are more elongated than haploid cells, and the characteristic shape (eccentricity) of the cells is unaffected by all environmental conditions and generation times tested. Mother cell volume increased during the cell cycle, although the pattern of this increase was affected by the environmental conditions. Under most growth conditions detectable mother cell volume increase occurred only during the budding phase, whereas under conditions of carbon limitation detectable increase only occurred during the unbudded phase. A consequence of this result is that the mean cell volume of haploids at bud initiation is relatively constant in all environments, including carbon limitation. This suggests that there is a critical size for bud initiation for haploids which is constant and independent of environmental conditions. The results for diploids are more complex. Coordination of growth and division in haploid cells can be explained by a simple model initially developed for prokaryotes by Donachie. A modification of this model is proposed to account for the results with diploids.     

MORPHOLOGY AND SYSTEMATICS OF SCIUROTHAMNION STEGENGAE GEN. ET SP. NOV. (CERAMIACEAE,RHODOPHYTA) FROM THE INDO‐PACIFIC1

A new ceramiaceous alga, Sciurothamnion stegengae De Clerck et Kraft, gen. et sp. nov., is described from the western Indian Ocean and the Philippines. Sciurothamnion appears related to the tribe Callithamnieae on the basis of the position and composition of its procarps and by the majority of post‐fertilization events. It differs, however, from all current members of the tribe by the presence of two periaxial cells bearing determinate laterals per axial cell. Additionally, unlike any present representative of the subfamily Callithamnioideae, no intercalary foot cell is formed after diploidization of the paired auxiliary cells. The genus is characterized by a terminal foot cell (“disposal cell”), which segregates the haploid nuclei of the diploidized auxiliary cell from the diploid zygote nucleus. The nature of three types of foot cells reported in the Ceramiaceae (intercalary foot cells containing only haploid nuclei, intercalary foot cells containing haploid nuclei and a diploid nucleus, and terminal foot cells containing only haploid nuclei) is discussed.     

Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae

The establishment of cell polarity was examined in the budding yeast, S. cerevisiae. The distribution of a polarized protein, the SPA2 protein, was followed throughout the yeast cell cycle using synchronized cells and cdc mutants. The SPA2 protein localizes to a patch at the presumptive bud site of G1 cells. Later it concentrates at the bud tip in budded cells. At cytokinesis, the SPA2 protein is at the neck between the mother and daughter cells. Analysis of unbudded haploid cells has suggested a series of events that occurs during G1. The SPA2 patch is established very early in G1, while the spindle pole body residues on the distal side of the nucleus. Later, microtubules emanating from the spindle pole body intersect the SPA2 crescent, and the nucleus probably rotates towards the SPA2 patch. By middle G1, most cells contain the SPB on the side of the nucleus proximal to the SPA2 patch, and a long extranuclear microtubule bundle intersects this patch. We suggest that a microtubule capture site exists in the SPA2 staining region that stabilizes the long microtubule bundle; this capture site may be responsible for rotation of the nucleus. Cells containing a polarized distribution of the SPA2 protein also possess a polarized distribution of actin spots in the same region, although the actin staining is much more diffuse. Moreover, cdc4 mutants, which form multiple buds at the restrictive temperature, exhibit simultaneous staining of the SPA2 protein and actin spots in a subset of the bud tips. spa2 mutants contain a polarized distribution of actin spots, and act1-1 and act1-2 mutants often contain a polarized distribution of the SPA2 protein suggesting that the SPA2 protein is not required for localization of the actin spots and the actin spots are not required for localization of the SPA2 protein. cdc24 mutants, which fail to form buds at the restrictive temperature, fail to exhibit polarized localization of the SPA2 protein and actin spots, indicating that the CDC24 protein is directly or indirectly responsible for controlling the polarity of these proteins. Based on the cell cycle distribution of the SPA2 protein, a "cytokinesis tag" model is proposed to explain the mechanism of the non-random positioning of bud sites in haploid yeast cells.     

Diploid Strains of the Pathogenic Basidiomycete Cryptococcus neoformans Are Thermally Dimorphic

Sia, R. A., Lengeler, K. B., and Heitman, J. 2000. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Cryptococcus neoformans is an opportunistic human pathogenic fungus with a defined sexual cycle. Clinical and environmental isolates of C. neoformans are haploid, and the diploid stage of the lifecycle is thought to be transient and unstable. In contrast, we find that diploid strains are readily obtained following genetic crosses of congenic MATα and MATa strains. At 37°C, the diploid strains grow as yeast cells with a single nucleus that is larger than a haploid nucleus, contains a 2n content of DNA by FACS analysis, and is heterozygous for the MATα and MATa loci. At 24°C, these diploid self-fertile strains filament and sporulate, producing recombinant haploid progeny in which meiotic segregation has occurred. In contrast to dikaryotic filament cells that are typically linked by fused clamp connections during mating, self-fertile diploid strains produce monokaryotic filament cells with unfused clamp connections. We also show that these diploid strains can be transformed and sporulated and that an integrated selectable marker segregates in a mendelian fashion. The diploid state could play novel roles in the lifecycle and virulence of the organism and can be exploited for the analysis of essential genes. Finally, the observation that dimorphism is thermally regulated suggests similarities between the lifecycle of C. neoformans and other thermally dimorphic human pathogenic fungi, including Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and Sporothrix schenkii.     

Die Verteilung der Chromosomen auf die Tochterzellkerne multipolarer Mitosen in euploiden Gewebekulturen von Microtus agrestis

In kidney epithelial cultures from female Microtus agrestis, 3,55% of all mitoses are multipolar, 94% of them tripolar. Feulgen photometric measurements of 21 tripolar mitoses reveal a total DNA amount corresponding to the mitotic diploid value (4c) in 5 cases, and to the tetraploid value (8c) in 16 cases, Diploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei each with a haploid DNA value (1c). Most tetraploid tripolar mitoses divide into one daughter nucleus with a diploid DNA value (2c) and two nuclei with a triploid DNA value (3c). Also the sex chromosomes are distributed to the daughter nuclei in the relation of 2∶3∶3. This can be seen in anaphase figures as well as in interphase nuclei presumably derived from tripolar mitoses, showing chromocenters according to the number of X-chromosomes. In two cases of tripolar tetraploid mitoses the resulting nuclei have a haploid, a triploid and a tetraploid DNA value. The DNA replication pattern is always identical in the daughter nuclei of diploid and tetraploid tripolar mitoses. — Our observations suggest segregation and distribution of haploid chromosome sets or multiples of haploid sets to the daughter nuclei of multipolar mitoses. They also show a possible way of formation of haploid and triploid cells in a basically diploid tissue. Presumably triploid nuclei (with 3 chromocenters) are capable of DNA synthesis.     

Caffeine enhancement of radiation killing in different strains of Saccharomyces cerevisiae.

Summary Haploid and diploid wild type strains, and three classes of radiation-sensitive mutants of Saccharomyces cerevisiae were tested for enhancement of UV-inactivation by caffeine in growth medium. In addition, the sensitizing effect of caffeine was studied in a haploid and a diploid wild type strain after gamma-irradiation. The drug sensitized the UV-irradiated cells of all strains except those reported to be only slightly UV-sensitive but highly sensitive to ionizing radiation. After gamma-irradiation, no caffeine-enhancement of killing was observed in stationary phase cells of either the haploid or the diploid strain. However, log-phase cells of both strains were partially sensitized.The results of both sets of experiments suggested that caffeine interferes with a recombinational repair occurring in cells in S or G2 phase.     

Nuclear division in the red algae Chondria nidifica and Chondria tenuissima

Cytological investigations are reported for two Chondria species, the Pacific species Chondria nidifica Harvey and Chondria tenuissima (Goodenough et Woodward) C. A. Agardh from the shore of the Marmara Sea in Istanbul. Nuclear division during mitosis and meiosis has been followed in somatic cells and in tetrasporangial mother cells respectively of diploid tetrasporic plants. The spherical interphase nucleus stains densely, showing many chromatin granules. Mitotic nuclei in the apical groove show a large number of chromosomes at metaphase; the chromosome number has been estimated at diakinesis to be 40 in both C. nidifica and C. tenuissima. The meiotic nuclei of tetraspore mother cells in prophase contain several relatively large nucleolar-derivatives in both species. The nucleolar derivatives disappear completely before the chromosomes begin to differentiate. In meiotic prophase the tetraspore mother cell enlarges from its original diameter. The period of the second meiotic anaphase seems to be extremely short in comparison with other nuclear phases. When the chromosomes reach the poles, they spread and subsequently form a relatively compact mass at telophase. The spindle has not been observed in C. tenuissima. Photographs are presented of nucleoli and nucleolar-derivatives in mitotic and meiotic divisions.     

Morphology,development and cytology of Taphrina maculans Butler

Morphology, development and nuclear behavior of the ascogenous stroma and asci in the infection spots have been described inTaphrina maculans Butler. The fungus forms subcuticular and intercellular mycelium in the leaf tissues and the ascogenous layers originate through division of the subcuticular hyphal cells in the infection sites. Germination of ascogenous cells starts with their elongation in the uppermost layer forming asci and ascospores without formation of stalk cells. Meiosis of the fusion (diploid) nucleus occurs in the young ascus as in otherTaphrina species devoid of stalk cells. The haploid chromosome complement in this species consists of 3 chromosomes (n=3). All the cells in the stromatic layer are potential ascogenous cells and ascus formation continues, until all of them are exhausted in the infection spot. Eight ascospores are normally formed in each ascus, but multi-plication of ascospores may occurin situ later. Three morphologically distinct types of ascus opening are encountered, which are apparently not correlated with prevalent environment. Multiplication of ascospores after their discharge from mature asci occurs by budding proceded by a mitotic division of the spore nucleus. Blastospores (budded cells) germinate into short hyphae and binucleate condition of cells originates by mitotic division of the nucleus. Occurrence of giant cells containing 2 nuclei is often observed. Possible origin of Uredinales fromTaphrina-like ancestors has been indicated due to their close resemblance.     

Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis

Summary In Saccharomyces cerevisiae, diploid strains which are respiratory deficient (e.g., rho^–) or are homozygous for the mating-type locus (i.e., either a/a or /) are unable to sporulate. In order to induce sporulation in these nonsporulating strains, the technique of protoplast fusion mediated by polyethylene glycol was adopted. In this study, the products of protoplast fusion were induced to sporulate without reversion to normal cells.Protoplasts from a respiratory-deficient diploid strain were mixed with those from a respiratory-competent haploid one carrying mitochondrial drug resistance markers, treated with 30% polyethylene glycol-4000 and 25 mM CaCl₂, and incubated in 0.1 M potassium acetate containing 0.8 M sorbitol as an osmotic stabilizer. After two days' incubation, asci with three to eight spores were formed at a frequency of 1×10^–3 to 2×10^–4. Sporulation was also observed in products of fusion between an a/a diploid and haploid strains and between an / diploid and a haploid strains. The analysis of the genotypes of spores revealed that when fusion products were cultured under conditions for sporulation, karyogamy did not take place, diploid nuclei underwent meiosis, and both diploid and haploid nuclei were able to develop into spores.     

A further study on chromosome minimization by protoplast fusion in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1–12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 μg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain.     

Subcellular localization of the interaction of bipolar landmarks Bud8p and Bud9p with Rax2p in Saccharomyces cerevisiae diploid cells

In Saccharomyces cerevisiae, the bud site selection of diploid cells is regulated by at least four persistent landmarks, Bud8p, Bud9p, Rax1p, and Rax2p. Bud8p and Bud9p are essential for the establishment of bipolar budding and localize mainly to the distal and the proximal poles, respectively. Their subcellular localizations are regulated through interaction with Rax1p/Rax2p. We investigated when and where Bud8p and Bud9p physically interact with Rax2p in vivo using a split-GFP method. GFP fluorescence showed that Bud8p physically interacted with Rax2p at the proximal or distal pole in unbudded cells; a physical interaction was also observed at the opposite pole to the growing bud in mother cells with a large-size bud. Bud9p physically interacted with Rax2p at the birth scar in budded mother cells. These observations suggest that the interaction of Rax2p with Bud8p and Bud9p may contribute to the translocation of bipolar landmarks to the correct sites.     

Flow cytometry reveals a high degree of genomic size variation and mixoploidy in various strains of the acellular slime mold Physarum polycephalum

High-resolution flow cytometry, using avian erythrocytes as an internal standard, was employed to study constitutive genome size variation of G2-phase nuclei of Physarum polycephalum strains during the macroplasmodial stage of their life cycle. Our results document a previously unknown extent of genome size variation and mixoploidy in this organism. The unimodal diploid strain Tu 291 displayed the largest genome of the strains tested; in contrast, the Colonia strain displayed only half of the Tu 291 G2-phase fluorescence, confirming its haploid nature. An additional strain, derived from a recent cross between Lu897 and Lu898 amoebae, must have arisen by selfing (propagation of only one of the parental genomes to the macroplasmodial stage), since its nuclei display close to the haploid G2-phase DNA content. The observation of a small fraction of corresponding diploid nuclei within the haploid population of this strain, while maintained as microplasmodia, supports the notion that meiosis in haploid strains may require the presence of diploid nuclei. Two of the descendants of the prototype haploid Colonia strain, which were kept for extended periods of time in submerse culture, proved to be near diploid and mixoploid. Polyploidization and subsequent loss of DNA thus seems to contribute to the extremes of genome size variation in Physarum. In addition to unimodal fluorescence distributions, a number of diploid strains displayed bi- and even trimodal distributions within harvests of a single G2-phase macroplasmodium. Analysis of these mixoploid strains by means of gaussian curve-fitting suggests that the smaller genome size differences in Physarum may arise in step-wise diminution of DNA in approximate units of 3-5% of the original Tu 291 genome.