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.     

Karyotype Characterization of In Vivo- and In Vitro-Derived Porcine Parthenogenetic Cell Lines

Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA embryo-derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.     

Evidence for dosage compensation in parthenogenetic Hymenoptera

Amount of DNA-Feulgen staining in individual somatic nuclei and mature sperm of the parthenogenetic wasps, Habrobracon juglandis, H. serinopae, and Mormoniella vitripennis, were determined with a scanning microdensitometer. The haploid genome for both species of Habrobracon was estimated to be 0.15–0.16×10^–12 g DNA, corresponding to a molecular weight of roughly 10×10¹⁰ daltons. The haploid genome of M. vitripennis is approximately twice this value, 0.33–0.34×10^–12 g, or about 20×10¹⁰ daltons. Measurements made on dividing nuclei from syncytial preblastoderm embryos of H. juglandis and M. vitripennis showed that the chromosomes of impaternate males were present in the haploid number and contained the C amount of DNA; whereas nuclei from female preblastoderm embryos contained the diploid number of chromosomes and the 2C amount of DNA. However, hemocyte and brain cell nuclei from either male or female adult wasps contained 2C and 4C amounts of DNA. Both sexes also showed equivalent levels of polyploidy (8C, 16C, or 32C) in Malpighian tubule nuclei. Therefore, in these parthenogenetic species, a mechanism must exist that compensates during later development for the initial two-fold difference in the chromatin content of somatic nuclei in haploid male and diploid female embryos. Hemocytes from impaternate Mormoniella diploid males and triploid females contain the 2C and 3C amounts of DNA, respectively. Therefore dosage compensation involves an additional cycle of DNA replication only in haploid cells, and it insures that a certain minimum quantity of DNA is received by each somatic cell.     

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.     

Cytochrome oxidase deficiency during development of amphibian nucleocytoplasmic hybrids.

The activity of mitochondrial cytochrome oxidase was investigated during the embryonic development of nucleocytoplasmic hybrids containing a nuclear genome derived from R. pipiens and a mitochondrial genome derived from R. palustris. Using a quantitative cytochemical approach, we found that the activity of cytochrome oxidase failed to increase during the development of these embryos. Control embryos containing a haploid chromosomal complement, derived from the same species as that from which the maternally inherited mitochondria is derived and hybrid crosses between R. palustris and R. pipiens, showed a significant increase in cytochrome oxidase activity during development. Oxygen uptake data from diploid and haploid R. pipiens embryos were in agreement with the data obtained by the cytochemical method. These results indicate that a normal pattern of cytochrome oxidase activity during embryonic development requires a nuclear genome which contains a haploid chromosomal complement derived from the same species as that from which the mitochondrial genome is derived.     

Genetics of Parthenogenesis in DROSOPHILA MELANOGASTER. I. the Modes of Diploidization in the Gynogenesis Induced by a Male-Sterile Mutant, ms(3)K81

Sperm that are produced by males homozygous for ms(3)K81 , a male sterile mutant of Drosophila melanogaster, are defective in syngamy but are capable of activating eggs to develop gynogenetically. The activated eggs usually produce haploid embryos, but a small fraction (10 ^-4–10^-5) of them give rise to diploid impaternate adults. To know the cytological mechanisms by which these impaternates restore diploidy, the genotypes of impaternate progeny obtained from females doubly heterozygous for visible markers were examined. The results show that, as generally found among parthenogenetic Drosophila, diploidy is restored after completing meiosis either by pronuclear fusion or by gamete duplication (doubling of a haploid cleavage nucleus). The fusion of two nonsister nuclei following meiosis II (central fusion) was indicated to be a predominant mode of diploidization in this species. Two meiotic mutants, mei-9 and mei-S332, which are known to greatly increase meiotic nondisjunction, did not cause an increased incidence of impaternates. This seems to exclude the possibility that some impaternates might have been derived from diploid egg nuclei produced through nondisjunction.     

The Chromosomes of Turkey Embryos during Early Stages of Parthenogenetic Development

In the early stages of parthenogenetic development in turkey eggs, many blastoderms are mosaics of haploid, diploid and polyploid cells. The genome composition of these blastoderms can be identified by C-banding. They may be generally described as either A-Z/2A-ZZ/nA-nZ or A-W/2A-WW/nA-nW and are found in a nearly 1:1 ratio. The blastoderms showing the W body (W⁺) become lethal within two days of incubation. The haploid cell proportion decreases rapidly during the early stage of development, and, as haploid cells decrease, the proportion of polyploid cells appears to increase. At six days of incubation, various kinds of parthenogenetic development can be observed. Their genome compositions are either diploid (2A-ZZ) or mosaic (A-Z/2A-ZZ). These findings suggest that diploid parthenogenesis occurs by either suppression of meiosis II or chromosome doubling some time after the first cleavage division. The frequent occurrence of mosaic blastoderms indicates that the majority, if not all, of the parthenogenetic embryos initiate their development in haploid ova.     

Haploid plants carrying a sodium azide-induced mutation (fdr1) produce fertile pollen grains due to first division restitution (FDR) in maize (Zea mays L.)

Key message

We induced a fdr1 mutation in maize which makes haploid plants male fertile due to first division restitution; the optimum sodium azide treatment on maize kernels has been identified.

Abstract

Sodium azide mutagenesis experiments were performed on haploid and diploid maize plants. Kernels with haploid embryos of maize inbred line B55 were induced by pollinating with RWS pollen. These kernels were treated with 0.2, 0.5, or 1.0 mM sodium azide solution for 2 h. The 0.5 mM solution was optimal for inducing numerous albino sectors on the treated plants without significant damage. Kernels of a maize hybrid, Oh43 × B55, were treated with sodium azide solutions at concentrations of 1.5, 2.0, 2.5, and 3.0 mM. Haploids were generated by pollinating RWS pollen. The highest rate of chlorophyll mutations in seedlings (15.3 % [13/85]) was recorded with the 2.5 mM concentration. A mutated haploid plant (PP1-50) with higher pollen fertility was isolated during the experiments. This haploid plant produced four kernels on the ear after selfing. These kernels were germinated and produced ears with full seed set after selfing. The haploid plants induced from PP1-50 diploids also exhibited high pollen fertility. In situ hybridization studies showed that meiocytes in PP1-50 haploid anthers underwent first division restitution at a rate of 48 % and produced equally divided dyads. We designated the genetic factor responsible for this high pollen fertility as fdr1. PP1-50 haploid ears exhibited high levels of sterility, as seen for regular haploids. Diploid PP1-50 meiocytes in the anther underwent normal meiosis, and all selfed progenies were normal diploids. We concluded that the fdr1 phenotype is only expressed in the anthers of haploid plants and not in the anthers of diploid plants.     

Locust embryos mosaic for haploid and diploid cells

Three embryos of Locusta migratoria are described which are mosaics of haploid and diploid cells. The chromosome constitution of the diploid cells discounts the possibility of a parthenogenetic origin. Polyspermy or polar body activation appear to be the most likely mechanisms of production.     

A Genetic Screen for Mutations Affecting Cell Division in the Arabidopsis thaliana Embryo Identifies Seven Loci Required for Cytokinesis

Cytokinesis in plants involves the formation of unique cellular structures such as the phragmoplast and the cell plate, both of which are required to divide the cell after nuclear division. In order to isolate genes that are involved in de novo cell wall formation, we performed a large-scale, microscope-based screen for Arabidopsis mutants that severely impair cytokinesis in the embryo. We recovered 35 mutations that form abnormally enlarged cells with multiple, often polyploid nuclei and incomplete cell walls. These mutants represent seven genes, four of which have previously been implicated in phragmoplast or cell plate function. Mutations in two loci show strongly reduced transmission through the haploid gametophytic generation. Molecular cloning of both corresponding genes reveals that one is represented by hypomorphic alleles of the kinesin-5 gene RADIALLY SWOLLEN 7 (homologous to tobacco kinesin-related protein TKRP125), and that the other gene corresponds to the Arabidopsis FUSED ortholog TWO-IN-ONE (originally identified based on its function in pollen development). No mutations that completely abolish the formation of cross walls in diploid cells were found. Our results support the idea that cytokinesis in the diploid and haploid generations involve similar mechanisms.     

Nuclear fusion in multinucleated giant cells during the sexual development of Dictyostelium discoideum

Macrocyst development in Dictyostelium discoideum, is generally considered a sexual phase. This development is initiated by the formation of a giant cell, the result of the fusion of two different mating type haploid cells, such as NC4 and HM1. The giant cell engulfs unfused surrounding cells to develop into a macrocyst. Therefore, if the macrocyst is a sexual structure, the giant cell must be a diploid zygote. However, under certain conditions, a very large multinucleated giant cell containing several dozens of nuclei is formed, followed by normal development into a macrocyst. In such a multinucleated giant cell, it was found that only two nuclei fuse together to produce a diploid zygote and all others disappear at the early stage of development. The diploid nucleus undergoes meiosis and subsequently subdivides into a number of haploid progeny cells later released from the macrocyst to initiate new life cycles.     

Haploidy influences Bak and Bcl-xL mRNA expression and increases incidence of apoptosis in porcine embryos

The objective of this study was to determine developmental pattern, total cell number, apoptosis and apoptosis-related gene expression in haploid and diploid embryos following parthenogenetic activation. In vitro-matured porcine oocytes were activated by electrical pulses and cultured in the absence or presence of cytochalasin B for 3 h. Zygotes with two polar bodies (haploid) and one polar body (diploid) were carefully selected and were further cultured in NCSU 23 medium containing 0.4% bovine serum albumin (BSA) for 7 days. The percentage of development to blastocyst stage was higher (p < 0.01) in the diploid than in the haploid parthenotes. In haploid blastocysts, average total cell number was significantly reduced (p < 0.05) and apoptosis was increased at day 7. The relative abundance of Bcl-xL and Bak mRNA in the diploid blastocysts was similar to that of in vivo-fertilized embryos. However, Bcl-xL was significantly decreased, and Bak mRNA was significantly increased (p < 0.05) in haploid parthenotes compared with the diploid parthenotes. These results suggest that the haploid state affects apoptosis-related gene expression which results in increased apoptosis and decreased developmental competence of haploid parthenotes.     

Evidence of the Insensitivity of the alpha-inc Allele to the Function of the Homothallic Genes in Saccharomyces Yeasts

The possible function of the α-inc allele (an α mating-type allele that is insensitive to the function of the homothallic gene system) was investigated by means of protoplast fusion. The fusion of protoplasts prepared from haploid strains of α-inc HO HMα HMa and α ho hmα HMa gave rise mainly to nonmating clones (58 of 64 isolates) and a few clones (six of 64 isolates) showing α mating type. Thirty of the 58 nonmating clones showed the diploid cell size and 28 clones had a larger cell size. Tetrad analysis of the nonmating clones with diploid cell size indicated that they were a/α-inc diploid; the normal α allele in α/α-inc cells was preferentially switched to an a allele. This observation further indicated that the HO/ho HMα/hmα HMa/HMa genotype is effective for the conversion of the α to a and that the inconvertibility of the α-inc allele is due to the insensitivity of the mating-type allele to the functional combination of the homothallic genes. It was suspected that fusion products larger than diploid cells might have been caused by multiple fusion of protoplasts.     

Induction of Phase Variation Events in the Life Cycle of the Marine Coccolithophorid Emiliania huxleyi

Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.     

Effects of haploidy and hybridization on the activities of four dehydrogenases in frog embryos

Total activities of four enzymes, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase, and 6-phosphogluconate dehydrogenase were measured in diploid and androgenetic haploid embryos resulting from crosses of Rana pipiens pipiens and Rana pipiens sphenocephala. Developmental curves of these enzyme activities were compared with the DNA content of the embryos. The results suggest that decreased total enzyme activity in abnormal androgenetic haploid hybrids is due to the effects of reduced cellular proliferation and cellular degradation caused by a general nucleocytoplasmic incompatibility, not to the factors directly affecting the synthesis of each enzyme.     

Haploid and diploid cell cultures from a haplo-diploid insect

Summary

This is the first report of haploid and diploid cell culture from the haplo-diploid parasitoid wasp, Mormoniella vitripennis. Cells were cultured from haploid and diploid wasps by collecting populations of eggs from virgin females (unfertilized, haploid, parthenogenetic eggs) and mated females (mostly fertilized, diploid eggs). Eggs were surface sterilized in 70% ethanol, followed by 50% Chlorox, and rinsed in phosphate buffered saline; larvae were allowed to hatch in culture. Larval cells were dissociated and cultured at 28°C in the presence of Grace's medium supplemented with fetal bovine serum. Most cells in the HMV (predominantly haploid) and DMV (predominantly diploid) cell cultures grew in suspension in the first week, formed monolayers of fibroblasts and epithelial cells by the second week in culture, and continued to grow in monolayers and vesicle-like structures for up to three months. Chromosome analysis of HMV. cells demonstrated over 70% haploid cells, with five chromosomes (N=5). The remainder were aneuploid. No diploid cells (2N= 10) were found in the HMV cell culture. Chromosome analysis of DMV cultures revealed 62% diploid, with ten chromosomes; 13% were haploid, with five chromosomes; the remainder were aneuploid. These data confirm that haploid and diploid cells can be cultured from a haplo-diploid insect species. The HMV cells which are predominantly haploid, and DMV cells which are predominantly diploid may be valuable models for the study of cellular and gene activity in haploid and diploid genetic milieux.     

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.     

Haplo-diploid locust embryos arising by accidental thelytoky in Chortoicetes terminifera investigated by G-banding

Two mosaic sibling embryos of the Australian plague locust, Chortoicetes terminifera are reported with haploid and diploid cell lines in widely differing proportions. One small chromosome pair involved in the two cases has alternative morphology and a B-chromosome is present in one. In addition, G-banding identifies two medium-sized chromosome pairs and alternative states of a second small pair. Using these markers it is clear that both diploid cell lines are homozygous for the chromosomes of the corresponding haploid line. These embryos have thus developed by accidental parthenogenesis from haploid cells, some of which were duplicated by endomitosis after development began.     

Recovery and development of parasexual fusion products inEnteromorpha linza (L.) J. Ag. (Ulvales,Chlorophyta)

Newly released zoospores fromEnteromorpha linza (L.) J. Ag. lack significant cellulose cell wall material and are suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca^{+ +}, PEG and centrifugation. Treated zoospores settled on glass cover slips within 3 h and were examined microscopically at 1000 ×. Presumptive fusion products were identified by their larger size and presence of twin chloroplasts and eyespots. Unfused zoospores adjacent to fusion cells were killed by 2–3 min exposure to blue light (410–490 nm) from a high pressure mercury illuminator. Unexposed fusion cells developed into uniseriate germlings within 10 days at which stage they could be readily identified at 60 × with a dissecting microscope and isolated by micropipette. Ten-day germlings from both unfused zoospores and fusion cells were stained with the DNA-localizing fluorochrome hydroethidine and relative nuclear DNA content determined with epi-(incident) UV illumination. All germlings were found to be uninucleate. Germlings from unfused zoospores had haploid nuclei with 1N = 10 and 1C and 2C levels of DNA, while germlings from fusion cells had diploid nuclei with 2N = 20 and 2C and 4C levels of DNA. These result are interpreted as evidence of karyogamy following parasexual zoospore fusions. Isolated diploid germlings, cultured for 10 weeks were found to conserve their 2N chromosome complements and elevated levels of nuclear DNA. Although most diploid germlings were morphologically similar to haploid control plants, some exhibited ‘gigas’ characteristics, including larger cells, chloroplasts, and nuclei. These results are discussed in terms of unique phenotypes that result when nuclear and organellar genes are combined in different ways.