Analysis of exchanges in differentially stained meiotic chromosomes of Locusta migratoria after BrdU-substitution and FPG staining

Differential staining of the sister-chromatids of meiotic chromosomes of Locusta migratoria was achieved following abdominal implantation of BrdU tablets and fluorescent plus Giemsa (FPG) staining of fixed and squashed testicular follicles. This paper presents a detailed analysis of crossover exchanges between light and dark chromatids in monochiasmate bivalents. Approximately half the bivalents studied had visible exchanges of dark and light chromatids associated with the chiasmata, as expected if chiasmata originate by breakage and rejoining exchange events between randomly selected non-sister chromatids. In all the bivalents studied the visible crossover exchanges coincided exactly with chiasmata thus showing that chiasma movement (terminalisation) does not occur subsequent to crossing-over in Locusta migratoria, and that chiasmata are therefore accurate indicators of crossing over. It was noted that a proportion (9.5%) of chiasmata were associated with apparently anomalous exchanges of dark and light chromatids which could not be explained by conventional crossing-over. Various hypotheses for the origin of these anomalous exchanges are considered.     

Chromatid structure: relationship between DNA content and nucleotide sequence diversity

Models of chromatid structure are based on inferences made from genetic, cytological, and cytochemical observations. An alternative approach can provide limits as to the number of identical subunits present in chromatids. This method is based on the demonstration that nucleotide sequence diversity may be estimated from the kinetics of renaturation of denatured DNA. Measurements of DNA content and renaturation rate constants are given for several eukaryotic DNAs. Control experiments involved measurements of renaturation kinetics of DNAs from bacteria and bacteriophage. These estimates show that most of the nucleotide sequences in mouse, Drosophila, and Ciona DNA are present only once per sperm. Since the reduction of DNA content during meiosis indicates that mouse sperm contain a haploid set of chromatids, it follows that a set of mouse meiotic chromatids contains a single copy of most sequences. Models of chromatid structure which postulate multiple subunits with identical nucleotide sequences are therefore not tenable for mouse meiotic chromatids. This method of analyzing nucleotide sequence diversity may be of general use in designing models of chromatid structure in other organisms.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation. Received: 3 July 1996 / Accepted: 4 October 1996     

Endomitosis in the mealy bug,Planococcus citri (Homoptera: Coccoidea)

Endomitosis in the Malpighian tubules of the mealy bug Planococcus citri (Risso) is described. The stages are identified on the basis of the length of the chromosomes and the distance between the sister chromatids or chromosomes. The appearance of the chromosomes in the various stages of endomitosis is compared to that in other hemipteran insects. During anaphase and telophase of endomitosis the ends of the sister chromatids and chromosomes tend to stay together longer than the other parts. It is suggested that in holokinetic chromosomes special regions for holding the chromatids together are concentrated near the ends of the chromosomes.Supported by grant GB1585 from the National Science Foundation, Washington, D.C.     

Meiosis in the male of Puto albicans (Coccoidea-Homoptera)

Cytologically, Puto has proved more primitive than its taxonomic position would indicate. In the single previously described example (Hughes-Schrader, 1944), an uncomplicated inverted meiotic sequence was described for the males. The present example, P. albicans, showed a significantly more primitive inverted sequence. Unlike the other examples reported for aphids and coccids, the chromatids of the dyads neither dissociated nor reassociated during interkinesis. Instead, they remained closely associated and interconnected by an unresolved terminal chiasmate attachment. At first metaphase, the spindle attachments were localized to a restricted region of the poleward surface of the chromatids. Localization of attachment during meiosis and close association of chromatids during interkinesis are both suggestive of similar but not identical conditions expected in ancestors with an uninverted meiotic sequence. A second species proved intermediate between P. albicans and that described by Hughes-Schrader in which a complete cycle of dissociation and reassociation occurred during interkinesis. In P. albicans the pachytene bivalents showed no structures suggestive of centric localizations. At their greatest condensation at first metaphase, the chromatids were clearly subdivided into half chromatids. Limited observations were made on chromosomes of other species of Puto and of Phenacoleachia zealandica, and also on spermiogenesis and mycetocyte formation in Puto. The discussion is devoted to considerations of chromatid subdivision, holokinetic chromosomes, and meiotic inversion and some evolutionary implications are mentioned.Supported by grant GB 4289 from the National Science Foundation.     

Genetic Control of Mitosis: Mutation ff3Causes Premature Beginning of Telophase Chromatin Reorganization in Drosophila melanogaster

The effect of cell cycle mutation ff3 on chromosome segregation was studied on fixed cells of neural ganglia of Drosophila melanogasterlarvae. The cell distributions by diameter of interphase nuclei and by distance between sister chromatid sets were compared at anaphase and telophase. In the control wild-type strain Lausenne, the cell distribution by distance between sister chromatids in anaphase was similar to their distribution by nuclear size. The mean distance between segregating chromatids at anaphase (l _av) coincided with the mean diameter of interphase nuclei (d _av) and was 8.3 m. Cells passed to telophase when chromatids were at least 10 m apart. The mutant ff3 strain differed from the control strain Lausenne in cell distribution by interphase nuclear diameter and distance between sister chromatids in anaphase; the mean nuclear diameter and mean distance between segregating chromatids similarly increased to 9.3 m. A specific feature of mitosis in mutant strain ff3 was a premature beginning of telophase chromatin reorganization. This caused the occurrence of cells with abnormally short (less then the interphase nuclear diameter) distance between sister chromatid sets in telophase but not in anaphase, as if these cells had passed from anaphase to telophase prematurely, during the chromatid movement toward poles in anaphase A.     

Steric hindrance and its effect on chromosome movement inVicia faba somatic cells

Summary The arrangement of chromosome arms in metaphases and anaphases has been studied inVicia faba root meristem cells. During metaphase, the long chromosome arms are aligned parallel to the spindle axis. As a consequence, at the onset of anaphase, one chromatid can move straight ahead to the spindle pole whereas the other has to invert its orientation. Specially in narrow cells it has been observed frequently that some chromatids move in a reverse orientation to the pole, i.e., they move telomere-first instead of centromere-first. This behaviour results in a chromatid which protrudes beyond the main group of late anaphase or telophase chromatids. It is dicussed that the most likely explanation for the phenomenon is that in narrow cells chromatid behaviour is influenced by steric hindrance by the tightly packed surrounding chromatids and microtubules. When there is insufficient room, some chromatids are unable to make the required U-turn. Under such conditions the kinetochore of a non-inverted chromatid pulls the chromatid in a reverse orientation to the pole. An alternative explanation, i.e., protruding chromatids being the result of a neocentric activity at the telomere end of a reverse-directed chromatid or the lateral associations of spindle microtubules, failed to find support by electron microscopical studies.     

Observations on dicentrics in living cells

Summary In previously irradiated endosperm cells of Haemanthus katherinae studied in vitro by means of micro-cinematography, two-kinetochore chromatids and dicentric chromosomes have been observed. Breaking of such dicentric chromatids and chromosomes has been analysed. Behaviour of some of the dicentric chromosomes during anaphase deserves special attention: interlocking dicentrics cut one through another and rejoin in a few minutes. In this way from a metaphase interlocking dicentric, two sister anaphase dicentrics are formed. Interlocked dicentrics can also uncoil and not break at all. In this case no activity was observed in one kinetochore of one dicentric in later stages of anaphase (two kinetochores were active in one dicentric and only one in its sister). Analysis of chromosome movements in two-kinetochore chromatids and dicentrics is also presented.     

Kinetochore and microtubules in two members of Chlorophyceae,Cladophora fracta and Spirogyra majuscula

Evidence is presented for the existence of a localised kinetochore with stratified fine structure in Cladophora and in Spirogyra. In the latter, there is the possibility of two kinetochores on the longer chromosomes. There is no evidence for a diffuse kinetochore. The nucleolus persists during mitosis in Cladophora on the nucleolar organising chromosomes, the granular material being lost from it very largely during metaphase and anaphase but the fibrillar material remaining. The persistent nucleolar material at metaphase and anaphase in Spirogyra is not attached to the nucleolar organising chromosomes but accumulates around all the chromosomes and chromatids, the microtubules of the spindle at anaphase passing through and possibly attaching to this nucleolar material and possibly assisting in the movement of the chromatids which are embedded within it.     

NUCLEAR BEHAVIOR IN THE VEGETATIVE HYPHAE OF CERATOCYSTIS FAGACEARUM

Vegetative nuclear division in Ceratocystis fagacearum (Bretz) Hunt was found to differ from classical mitosis in that: (1) division always occurs perpendicular to the longitudinal axis of the cell, (2) anaphase movement is unilateral and unsynchronized, (3) a spindle occurs only between separating chromatids. Interphase and prophase nuclei and nucleoli are morphologically similar to those in higher plants. At metaphase the associated chromosomes form a bar of chromatin and lie against the hyphal wall. Spindle fibers appear between separating chromatids, perhaps pushing them apart. When nuclear division is complete the nuclei become attenuated and migrate. Vegetative nuclear division in C. fagacearum may be an evolutionary form of classical mitosis.     

Mitotic instability in wheat×Thinopyrum ponticum derivatives revealed by chromosome counting, nuclear DNA content and histone H3 phosphorylation pattern

To evaluate the mitotic stability of Triticum aestivum×Thinopyrum ponticum derivatives (BC₂F₇ and BC₂F₅ doubled haploids), chromosome counting by both conventional and immunostaining techniques, and measurement of DNA content were performed. The wheat progenitor line, PF 839197, the wheat recurrent parent CEP 19 and the control Chinese Spring were also investigated. In the hybrid derivatives, chromosome number ranged from 2n=36 to 60, with a predominance of chromosome numbers higher than 2n=42, that was confirmed by determination of nuclear DNA content. Chinese Spring and PF 839197 were stable, but CEP 19 showed chromosome number variation (20%). Analyses of non-pretreated cells revealed the presence of anaphase bridges, lagging chromatids, chromosome fragments and micronuclei. Immunostaining with an antibody recognizing histone H3 phosphorylated showed dicentric chromatids forming anaphase bridges and pericentromeric phosphorylation at centric chromosome fragments but not at lagging chromatids. The possible causes of the observed mitotic instability are discussed.     

Spermatogenesis in the aphid Amphorophora tuberculata (Homoptera,Aphididae)

Spermatogenesis was studied in Amphorophora tuberculata Brown & Blackman, a species of aphid with n=2. Spermatogonia have 2=3 (AA+XO). In early prophase I the autosomal homologues are united terminally to form a tandem bivalent. No evidence could be found of synapsis or of the formation and terminalisation of chiasmata. The terminal connection of the autosomes is retained until late in prophase II. Sister chromatids separate, and autosomal half-bivalents move apart at anaphase I, but the division is incomplete, the X chromosome forming a thin chromatin bridge between the two autosomal half-bivalents. In prophase II the autosomal half-bivalents double back on themselves, so that non-sister chromatids become aligned in parallel. The X chromosome then becomes associated with one of the autosomal half-bivalents. Anaphase II separates the non-sister chromatids, and meiosis is thus post-reductional.     

Genetic Control of Mitosis: Features of Anaphase Separation of Sister Chromatid Sets in Mutant Strain aar V158 in Drosophila melanogaster

The effect of mutation aar ^V158 on anaphase separation of chromatids was studied on fixed cells of neural ganglia of Drosophila melanogaster larvae. It was shown that mutation aar ^V158 causes three types of defective chromosome segregation manifested as (1) monopolar anaphase, (2) separation of chromatids to an abnormally short distance in anaphase, and (3) bridging and lagging of some chromatids or prolonged asynchronous separation of sister chromatid sets to the poles in anaphase. We believe that the former two types of defective segregation are caused by disturbed centrosome separation at the beginning of mitosis and the third type, by defects in chromatid separation during anaphase. During the two-year maintenance of the mutation in a heterozygous state, partial correction (adaptive modification) of the defects of type 1 and type 2 (but not type 3) occurred. The correction of type 1 and type 2 defects during adaptogenesis depended on the genotype: in heterozygotes and homozygotes, respectively type 1 and type 2 were preferentially corrected. The frequency of type 3 defects remained constant during the two-year period of maintenance of the mutation in a heterozygous state. However, in all variants of the experiment, their frequency decreased with increasing distance between the sister chromatid sets. In the cells that completed the previous division with abnormalities, the checkpoint system is supposed to effectively arrest the cell cycle in the subsequent division.     

The mitotic centromeric protein MEI-S332 and its role in sister-chromatid cohesion

Faithful segregation of sister chromatids during cell division requires properly regulated cohesion between the sister centromeres. The sister chromatids are attached along their lengths, but particularly tightly in the centromeric regions. Therefore specific cohesion proteins may be needed at the centromere. Here we show that Drosophila MEI-S332 protein localizes to mitotic metaphase centromeres. Both overexpression and mutation of MEI-S332 increase the number of apoptotic cells. In mei-S332 mutants the ratio of metaphase to anaphase figures is lower than wild type, but it is higher if MEI-S332 is overexpressed. In chromosomal squashes centromeric attachments appear weaker in mei-S332 mutants than wild type and tighter when MEI-S332 is overexpressed. These results are consistent with MEI-S332 contributing to centromeric sister-chromatid cohesion in a dose-dependent manner. MEI-S332 is the first member identified of a predicted class of centromeric proteins that maintain centromeric cohesion. Received: 11 December 1998; in revised form: 4 August 1999 / Accepted: 13 August 1999     

Identification of the Meiotic Division of Malarial Parasites

Zygotes of Plasmodium berghei were cultured 15–25 h in vitro to yield mature infective ookinetes. Samples taken in the first 5 h of culture were examined by electron microscopy. Meiotic figures were detected in the nuclei of the zygotes. Threadlike leptotene chromatids (chromosomes) condensed from attachment plaques on the nuclear envelope; chromatid pairing followed (zygotene), with synaptonemal complexes subsequently appearing (pachytene). These complexes persisted into metaphase but dissociated when the chromatids rapidly decondensed during anaphase. At telophase of the first meiotic division the kinetochores were retracted toward two small spindle complexes, which were found at widely separated poles in the nuclear envelope. The observations are consistent with a haploid genome of 8–10 chromosomes.     

GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion

Background Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy.Results We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)–Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP–Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate.Conclusions We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.     

Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach

Kinetochores and chromatid cores of meiotic chromosomes of the grasshopper species Arcyptera fusca and Eyprepocnemis plorans were differentially silver stained to analyse the possible involvement of both structures in chromatid cohesiveness and meiotic chromosome segregation. Special attention was paid to the behaviour of these structures in the univalent sex chromosome, and in B univalents with different orientations during the first meiotic division. It was observed that while sister chromatid of univalents are associated at metaphase I, chromatid cores are individualised independently of their orientation. We think that cohesive proteins on the inner surface of sister chromatids, and not the chromatid cores, are involved in the chromatid cohesiveness that maintains associated sister chromatids of bivalents and univalents until anaphase I. At anaphase I sister chromatids of amphitelically oriented B univalents or spontaneous autosomal univalents separate but do not reach the poles because they remain connected at the centromere by a long strand which can be visualized by silver staining, that joins stretched sister kinetochores. This strand is normally observed between sister kinetochores of half-bivalents at metaphase II and early anaphase II. We suggest that certain centromere proteins that form the silver-stainable strand assure chromosome integrity until metaphase II. These cohesive centromere proteins would be released or modified during anaphase II to allow normal chromatid segregation. Failure of this process during the first meiotic division could lead to the lagging of amphitelically oriented univalents. Based on our results we propose a model of meiotic chromosome segregation. During mitosis the cohesive proteins located at the centromere and chromosome arms are released during the same cellular division. During meiosis those proteins must be sequentially inactivated, i.e. those situated on the inner surface of the chromatids must be eliminated during the first meiotic division while those located at the centromere must be released during the second meiotic division.by D.P. Bazett-Jones     

Cohesion and centromere activity are required for phosphorylation of histone H3 in maize

Haspin‐mediated phosphorylation of histone H3 at threonine 3 (H3T3ph) promotes proper deposition of Aurora B at the inner centromere to ensure faithful chromosome segregation in metazoans. However, the function of H3T3ph remains relatively unexplored in plants. Here, we show that in maize (Zea mays L.) mitotic cells, H3T3ph is concentrated at pericentromeric and centromeric regions. Additional weak H3T3ph signals occur between cohered sister chromatids at prometaphase. Immunostaining on dicentric chromosomes reveals that an inactive centromere cannot maintain H3T3ph at metaphase, indicating that a functional centromere is required for H3T3 phosphorylation. H3T3ph locates at a newly formed centromeric region that lacks detectable CentC sequences and strongly reduced CRM and ZmBs repeat sequences at metaphase II. These results suggest that centromeric localization of H3T3ph is not dependent on centromeric sequences. In maize meiocytes, H3T3 phosphorylation occurs at the late diakinesis and extends to the entire chromosome at metaphase I, but is exclusively limited to the centromere at metaphase II. The H3T3ph signals are absent in the afd1 (absence of first division) and sgo1 (shugoshin) mutants during meiosis II when the sister chromatids exhibit random distribution. Further, we show that H3T3ph is mainly located at the pericentromere during meiotic prophase II but is restricted to the inner centromere at metaphase II. We propose that this relocation of H3T3ph depends on tension at the centromere and is required to promote bi‐orientation of sister chromatids.     

On the mechanism of differential giemsa staining of BrdU-substituted chromatids

This paper analyses the effect of acid hydrolysis on the differential Giemsa staining of 5-bromo-2deoxyuridine (BrdU) substituted chromatids in human and plant chromosomes, after treatment with a fluorochrome and light. Human lymphocytes and Allium cepa L. root tips were grown in BrdU for two or three cell cycles. Lymphocyte spreadings and meristem squashes were treated with fluorochrome Hoechst 33258, exposed to sunlight, hydrolysed with 5N HCl and stained with Giemsa. This acid hydrolysis improves the differential staining of BrdU substituted and non-substituted chromatin. It also allows the differentiation of sister chromatids with the DNA specific dye azure-A.     

Chromosome cohesion decreases in human eggs with advanced maternal age

Aneuploidy in human eggs increases with maternal age and can result in infertility, miscarriages, and birth defects. The molecular mechanisms leading to aneuploidy, however, are largely unknown especially in the human where eggs are exceedingly rare and precious. We obtained human eggs from subjects ranging from 16.4 to 49.7 years old following in vitro maturation of oocyte‐cumulus complexes isolated directly from surgically removed ovarian tissue. A subset of these eggs was used to investigate how age‐associated aneuploidy occurs in the human. The inter‐kinetochore distance between sister chromatids increased significantly with maternal age, indicating weakened cohesion. Moreover, we observed unpaired sister chromatids from females of advanced age. We conclude that loss of cohesion with increasing maternal age likely contributes to the well‐documented increased incidence of aneuploidy.