WHAT DO WE KNOW ABOUT CHAROPHYTE (STREPTOPHYTA) LIFE CYCLES?1

The charophyte algae are the closest living relatives of land plants. Their life cycles are usually characterized as haploid with zygotic meiosis. This conclusion, however, is based on a small number of observations and on theoretical assumptions about what kinds of life cycle are possible. Little is known about the life cycles of most charophytes, but unusual phenomena have been reported in comparatively well‐studied taxa: Spirogyra and Sirogonium are reported to produce diploid gametes with synapsis of homologous chromosomes before fusion of gametic nuclei; Closterium ehrenbergii is reported to undergo chromosome reduction both before and after syngamy; and zygotes of Coleochaete scutata are reported to replicate their DNA to high levels before a series of reduction divisions. All of these phenomena require confirmation, as does the conventional account.     

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.     

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.     

Allelic and Ectopic Interactions in Recombination-Defective Yeast Strains

Ectopic recombination in the yeast Saccharomyces cerevisiae has been investigated by examining the effects of mutations known to alter allelic recombination frequencies. A haploid yeast strain disomic for chromosome III was constructed in which allelic recombination can be monitored using leu2 heteroalleles on chromosome III and ectopic recombination can be monitored using ura3 heteroalleles on chromosomes V and II. This strain contains the spo13-1 mutation which permits haploid strains to successfully complete meiosis and which rescues many recombination-defective mutants from the associated meiotic lethality. Mutations in the genes RAD50, SPO11 and HOP1 were introduced individually into this disomic strain using transformation procedures. Mitotic and meiotic comparisons of each mutant strain with the wild-type parental strain has shown that the mutation in question has comparable effects on ectopic and allelic recombination. Similar results have been obtained using diploid strains constructed by mating MATa and MAT alpha haploid derivatives of each of the disomic strains. These data demonstrate that ectopic and allelic recombination are affected by the same gene products and suggest that the two types of recombination are mechanistically similar. In addition, the comparison of disomic and diploid strains indicates that the presence of a chromosome pairing partner during meiosis does not affect the frequency of ectopic recombination events involving nonhomologous chromosomes.     

Genetic analysis of polyauxotrophy and early mitotic progeny of Saccharomyces cerevisiae zygotes. II. Spore-forming segregants in the mitotic and meiotic progeny of polyauxotrophic clones

This article continues the investigation of polyauxotrophic (PA) clones formed in early mitotic progeny of zygotes. Cloning and segregation analysis of PA progeny suggest an unusual state of diploid genome in these strains, which is expressed as elimination of the dominance effect of the wild allele and as suppression or conversion of either of two loci of mating type. In PA progeny, except for recombinant haploids, sporulating diploids and unstable clones were detected. The tetrad analysis of the diploids points to homozygotization for individual markers. Over-replication of diploid set of chromosomes, prior to meiosis, and replacement of the haploid nucleus (the product of meiosis) for the diploid nucleus may explain the appearance of sporulating segregants in the diploid meiotic progeny. Unstable segregants may be considered as heterokaryons with complex interaction of nuclei.     

ALTERNATION OF GENERATIONS IN ALGAE: ITS COMPLEXITY, MAINTENANCE AND EVOLUTION

Life histories of photosynthetic eukaryotes traditionally-termed algae exhibit a considerably greater degree of complexity than those of ‘higher cryptogams.’ Some algae have a so-called ‘obligate’alternation between spore-producing and gamete-producing phases, but the majority seem capable of following other pathways depending upon environmental conditions. In only four algal classes do life histories show a change in morphological and/or nuclear phases. The following basic life histories are recognized in the Chlorophyceae, Phaeophyceae and Rhodophyceae:(a) monophasic, a diploid or haploid phase, (b) two or more phases, most commonly an alternation of an isomorphic or heteromorphic haploid gametangial phase and a diploid sporangial phase, and (c) three phases (unique to florideophyte Rhodophyceae), with a diploid spore-producing phase (carposporophyte) developing on the gametangial phase, a diploid phase (tetrasporophyte if meiosis is sporic) and a haploid gametangial phase. Evidence from recent research indicates that in many algae there is an uncoupling of the morphological and nuclear phases. The dominance of one phase and suppression of another has been suggested to be due to the common occurrence in algae of apogamy, apomeiosis and parthenogenesis. Free-living morphs in heteromorphic life histories may be morphologically so dissimilar that formerly they were attributed to different genera. Evolution of the carposporangial phase in red algae is speculated to be a means of achieving zygotic amplification to compensate for the infrequency of syngamy. Such amplification allows the production of a large number of dispersible products from a single fertilization. The direct development of a free-living tetrasporangial phase is considered another mechanism for achieving amplification. In freshwater red algae the growth of an upright phase from a perennial microscopic one is considered an adaptation for maintaining their upstream position. Life history pathways in algae are controlled by subtle environmental influences (e.g. photoperiodism, temperature, light quality, nutrients). Experimental evidence is lacking to support the contention that spatial and/or temporal partitioning of the environment is a mechanism favouring the maintenance of heteromorphy. Herbivory is known to be an important selective force suppressing some morphs and accentuating the seasonal dominance of others. Differential resistance of morphs to herbivory in environments where grazing intensity is predictable may lead to the selective maintenance of heteromorphy. Algal life history patterns are unexplored in terms of evolutionary processes. Various models for the evolution of biphasic or polyphasic life histories stress the importance of the capacity for both asexual dispersal of successful genotypes and for the generation of new genotypes via meiosis and syngamy. All evidence points to the fact that many life history processes operative in algae differ significantly from those described for other cryptogams.     

A comparison of in vitro flowers to in vivo flowers of haploid and diploidNicotiana tabacum L.

Thin cell layers (TCLs) were cultured from inflorescences of diploid (2n=4x=48) and haploid (2n=2x=24)Nicotiana tabacum L. "Samsun" and the subsequent flowers formed in vitro were then compared to in vivo flowers. Plants derived from TCLs possessed flowers that were typical of their seed or androgenetically-derived counterparts, whereas de novo flowers from TCLs were abnormal when compared to their counterparts. The TCLs of haploid plants produced more flower buds than diploid TCLs, and did so in a shorter period of time. In vitro flowers and anthers at both ploidy levels were considerably smaller than the in vivo flowers; in vitro flowers also had variable numbers of anthers and pistils. The embryogenic capacity of anthers taken from in vivo diploid flowers was 5 times greater than that of in vitro diploid or haploid anthers. In vivo haploid anthers produced no embryoids, whereas in vitro haploid anthers did produce embryoids. Observations of mitotic cells in root tips of plants derived from anther cultures of in vitro haploid flowers revealed a mixoploid nature. Diploid meiosis was regular and haploid meiosis was irregular regardless of the origin (in vitro or in vivo) of the flowers.Supported by state Hatch funds.     

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.     

The meiotic behaviour of inversions in polyploid aloineae

In addition to the four classical inversion phenomena, meiosis in tetraploid paracentric inversion heterozygotes produces multiple dicentric and complex tricentric bridges which were previously little understood. Also formed are open loop chromatids which can give rise to dicentric chromosomes in the progeny. A qualitative and quantitative study of the first and second meiotic division in Gasteria nigricans var. crassifolia (Liliaceae, Aloineae) agrees closely with theoretical considerations. Breakage of dicentric bridges results in the formation of chromosomes carrying large terminal deletions. These are shown to be viable in the diploid gametes produced by tetraploids because of the buffering effect of the second haploid set of chromosomes.     

Unequal sister chromatid exchange in the rDNA array of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the nucleolar organiser region (NOR) is located on chromosome XII. It contains 100-200 copies of rDNA--a minimum of 20 rDNA genes in tandem--and is termed the RDN locus. Yeast cells may exist in either haploid or diploid form. There are two forms of life cycle: haploid and diploid cells double by mitosis, and diploid cells are reduced to the haploid state by meiosis. Diploid cells have two homologous chromosomes for each of the 16 chromosomes. They are usually of the same size. However, in this study it is shown that homologous chromosomes XII can become different in size due to unequal sister chromatid exchange during mitosis in 'old' cells.     

High-throughput knockout screen in Schizosaccharomyces pombe identifies a novel gene required for efficient homolog disjunction during meiosis I

Meiosis is the process which produces haploid gametes from diploid precursor cells. This reduction of chromosome number is achieved by two successive divisions. Whereas homologs segregate during meiosis I, sister chromatids segregate during meiosis II. To identify novel proteins required for proper segregation of chromosomes during meiosis, we applied a high-throughput knockout technique to delete 87 S. pombe genes whose expression is upregulated during meiosis and analyzed the mutant phenotypes. Using this approach, we identified a new protein, Dil1, which is required to prevent meiosis I homolog non-disjunction. We show that Dil1 acts in the dynein pathway to promote oscillatory nuclear movement during meiosis.     

Repair of DNA double-strand breaks requires two homologous DNA duplexes

DNA repair and cell survival in haploid and its diploid derivative strains ofSaccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in theG ₁ stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.     

Karyotype Variability in Yeast Caused by Nonallelic Recombination in Haploid Meiosis

Chromosomes of altered size were found in the meiotic products of a haploid Saccharomyces cerevisiae strain by pulsed field gel electrophoretic separation of whole chromosomes. About 7% of haploid meioses produced chromosomes that differed by >/=10 kb from their wild-type counterparts. Chromosomes most often became enlarged or shortened due to recombination events between sister chromatids at nonallelic sequences. By this mechanism chromosome III acquired tandem arrays of up to eight extra copies of the ~100 kb MAT-HMR segment during repeated rounds of haploid meioses. Enlarged chromosomes III were unstable and changed their size during meiosis more often than remaining unchanged. Altered chromosomes appeared also as the products of intrachromatid recombination and of reciprocal translocations caused by ectopic recombination between nonhomologous chromosomes. In diploid meiosis, chromosomes of altered size occurred at least 10 times less frequently, whereas in mitotic cultures cells with altered karyotypes were virtually absent. The results show that various forms of ectopic recombination are promoted by the absence of allelic homologies.     

Haploid Meiosis in Arabidopsis: Double-Strand Breaks Are Formed and Repaired but Without Synapsis and Crossovers

Two hallmark features of meiosis are i) the formation of crossovers (COs) between homologs and ii) the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype) in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Un ménage à quatre: the molecular biology of chromosome segregation in meiosis

Sexually reproducing organisms rely on the precise reduction of chromosome number during a specialized cell division called meiosis. Whereas mitosis produces diploid daughter cells from diploid cells, meiosis generates haploid gametes from diploid precursors. The molecular mechanisms controlling chromosome transmission during both divisions have started to be delineated. This review focuses on the four fundamental differences between mitotic and meiotic chromosome segregation that allow the ordered reduction of chromosome number in meiosis: (1) reciprocal recombination and formation of chiasmata between homologous chromosomes, (2) suppression of sister kinetochore biorientation, (3) protection of centromeric cohesion, and (4) inhibition of DNA replication between the two meiotic divisions.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.     

Automictic parthenogenesis in the stick insectBacillus rossius Rossi (Cheleutoptera,phasmidae)

The process which restores the zygoid number of chromosomes in the thelytokously parthenogenetic stick insectBacillus rossius Rossi, is described. During the first 15 days after oviposition approximately 200 haploid (A+X=18 chromosomes) pre-blastoderm cells develop in the egg-cortex. During the second period of 15 days up to about 2000 blastoderm cells develop in which were found: haploid mitoses, C-mitoses (A+X=18 chromosomes), endomitoses, diploid mitoses (2A+2X=36 chromosomes) and pycnosis. The next 10 days the number of pycnotic nuclei decreases gradually and the blastoderm cells multiply by diploid mitosis only. Thereafter, a diploid germ band is formed either directly or after a diapause. It is concluded that C-mitosis in haploid cells restores the somatic number of chromosomes (automictic parthenogenesis), that a portion of the cells in C-mitosis degenerates and that endomitosis leads to the formation of some polyploid cells only. This development is discussed in relation to the occurrence of geographic parthenogenesis and of impaternate males.     

Nullisomic Tetrahymena. II. a Set of Nullisomics Define the Germinal Chromosomes

Crosses of a diploid Tetrahymena thermophila to a strain with a haploid germinal nucleus result in chromosome loss during meiosis in the haploid. The resulting monosomics can be made nullisomic by a special cross that induces homozygosis of a meiotic product of the germinal nucleus, but retention of the parental somatic nucleus. The creation and testing of single nullisomics for three of the five chromosome pairs and a triple nullisomic missing another pair is presented. Taken together, these strains make possible a series of crosses in which all but one of the chromosomes is missing in one parent. This set of nullisomics can, therefore, be used to map any mutation in Tetrahymena to a specific chromosome.     

Chromosome spatial order in human cells: evidence for early origin and faithful propagation

We have investigated the origin and nature of chromosome spatial order in human cells by analyzing and comparing chromosome distribution patterns of normal cells with cells showing specific chromosome numerical anomalies known to arise early in development. Results show that all chromosomes in normal diploid cells, triploid cells and in cells exhibiting nondisjunction trisomy 21 are incorporated into a single, radial array (rosette) throughout mitosis. Analysis of cells using fluorescence in situ hybridization, digital imaging and computer-assisted image analysis suggests that chromosomes within rosettes are segregated into tandemly linked “haploid sets” containing 23 chromosomes each. In cells exhibiting nondisjunction trisomy 21, the distribution of chromosome 21 homologs in rosettes was such that two of the three homologs were closely juxtaposed, a pattern consistent with our current understanding of the mechanism of chromosomal nondisjunction. Rosettes of cells derived from triploid individuals contained chromosomes segregated into three, tandemly linked haploid sets in which chromosome spatial order was preserved, but with chromosome positional order in one haploid set inverted with respect to the other two sets. The spatial separation of homologs in triploid cells was chromosome specific, providing evidence that chromosomes occupy preferred positions within the haploid sets. Since both triploidy and nondisjunction trisomy 21 are chromosome numerical anomalies that arise extremely early in development (e.g., during meiosis or during the first few mitoses), our results support the idea that normal and abnormal chromosome distribution patterns in mitotic human cells are established early in development, and are propagated faithfully by mitosis throughout development and into adult life. Furthermore, our observations suggest that segregation of chromosome homologs into two haploid sets in normal diploid cells is a remnant of fertilization and, in normal diploid cells, reflects segregation of maternal and paternal chromosomes. Received: 19 January 1998; in revised form: 28 May 1998 / Accepted: 30 June 1998