Pachytene mapping of the C 9 and acrocentric bivalents in the human oocyte

Summary Provisional maps are presented for all acrocentric bivalents and bivalent 9, according to their chromomere patterns at pachytene in the human oocyte. Each G band is subdivided into several sub-bands whose number varies according to the degree of chromosomal compacting. Chromomere number and sequence are in basic agreement with those observed in late prophase mitotic chromosomes. Thus, metaphase G bands of mitotic chromosomes result from progressive compressing together of smaller chromomeres whose individuality disappears as chromosomal condensation increases with progression of prophase.     

Meiosis of trisomy 21 in the human pachytene oocyte

Association modalities of the three 21 chromosomes were studied during pachytene in three trisomy 21 fetuses whose chromosomal constitution was identified following amniocentesis. -- Three classes of images were observed: a trivalent, a trivalent presenting an important asynaptic region of the long arm, and a bivalent accompanied by a univalent. Such behaviour is analagous to that observed in all trisomic organisms. -- We have been able to establish the sequence of chromomeres, whose number varies from 9 to 14 according to the state of contraction in the 21 chromosome. Each band is thus subdivided into several sub-bands: at maximal elongation 2 sub-bands for band p11, 4 for q21 and 3 for q222. In addition, the interchromomeric clear bands q221 and q223 are also subdivided by the presence of a very small chromomere. In this way, the G-bands visible on mitotic metaphase chromosomes result from the compression together of several chromomeres whose individuality disappears as chromosomal condensation increases with progression of prophase.     

Chromosome condensation in mitosis and meiosis of rye (Secale cereale L.)

Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids.     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.     

Mechanisms of chromosome banding : VI. Whole mount electron microscopy of banded metaphase chromosomes and a comparison with pachytene chromosomes

Chinese hamster chromosomes, banded by exposure to actinomycin D during the G 2 period, were examined by whole mount electron microscopy. Bands of condensed chromatin were present in unstained preparations that were not fixed with methanol-acetic acid indicating that the differential condensation of chromatin plays a role in banding by this technique. There was a tendency for interdigitation of the chromatin of the homologous bands on sister chromatids. Since previous studies had shown that the bands of mitotic chromosomes matched the chromomeres of meiotic chromosomes, whole mount electron microscope preparations of pachytene chromosomes were also examined. These suggest that in addition to condensation the chromatin of the chromomeres may also have a higher density of attachment sites to the lateral element of the synaptonemal complex, and probably to the nuclear membrane in interphase cells.     

A new chromosome model

We present a new model of the three-dimensional structure of chromosomes. With DNA and protein staining it could be shown by high-resolution scanning electron microscopy that metaphase chromosomes are mainly composed of DNA packed in "chromomeres" (coiled solenoides) and a dynamic matrix formed of parallel protein fibers. In the centromeric region, the chromomeres are less densely packed, giving insight into the matrix fibers. We postulate that chromosome condensation is achieved by the binding of solenoids to matrix fibers which have contact sites to one another and move antiparallel to each other. As condensation progresses, loops of solenoids accumulate to form additional chromomeres, causing chromosomes to become successively shorter and thicker as more chromomeres are formed. For sterical reasons, a tension vertical to the axial direction forces the chromatids apart. The model can simply explain the enormous variety of chromosome morphology in plant and animal systems by varying only a few cytological parameters. Primary and secondary constrictions and deletions are defined as regions devoid of chromomeres. Even in the highly condensed metaphase, all genes would be easily accessible.     

Changes in the organization of chromosomes during the cell cycle: response to ultraviolet light.

Fusion between mitotic and interphase cells results in the premature condensation of the interphase chromosomes into a morphology related to the position in the cell cycle at the time of fusion. These prematurely condensed chromosomes (PCC) have been used in conjunction with u.v. irradiation to examine the interphase chromosome condensation cycle of HeLa cells. The following observations have been made: (I) There is a progressive decondensation of the chromosomes during G1 which is accentuated by u.v. irradiation: (2) The chromosomes become more resistant to u.v.-induced decondensation during G2 and mitosis. (3) There is a close correlation between the degree of chromosome decondensation and the amount of unscheduled DNA synthesis induced by u.v. irradiation during G1 and mitosis: (4) Hydroxyurea enhances the ability of u.v. irradiation to promote the decondensation of chromosomes during G1, G2 and mitosis. Hydroxyurea also potentiates the lethal action of u.v. irradiation during mitosis and G1. These data are discussed in relation to the suggestion that chromosomes undergo a progressive decondensation during G1 and condensation during G2.     

Teniposide, a topoisomerase II inhibitor, prevents chromosome condensation and separation but not decondensation in fertilized surf clam (Spisula solidissima) oocytes

DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized Spisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin.     

Nuclear asynchrony in multinucleate rat kangaroo cells

Multinucleate (MN) cells were induced in PtK1 cells by colcemid treatment. A large percentage of cells developed nuclear asynchrony both in relation to DNA synthesis and mitosis within one cell cycle. Asynchrony could be traced even in metaphase and anaphase cells in which interphase nuclei, PCC of S-phase nuclei and less condensed prophase-like chromosomes could be observed along with normally condensed chromosomes. The occurrence of such abnormalities in these large MN cells may be explained on the basis of an uneven distribution of inducer molecules of DNA synthesis and mitosis due to cytoplasmic compartmentation. The less condensed form of all the chromosomes except chromosome 4 could be traced in asynchronous metaphase. The failure of the less condensed chromosomes to undergo complete condensation does not always appear to result from late entry of nuclei containing these chromosomes into G2 phase. It is likely that chromosome 4 carries gene(s) for chromosome condensation, as this chromosome itself never appears in a less condensed form. The inducers for chromosome condensation may not always be available at equal concentrations to all chromosomes located in separate nuclei, thus they may sometimes fail to undergo complete condensation before other nuclei reach the end of prophase, when the nuclear envelopes of all nuclei present in the cell break down simultaneously.     

Chromosome condensation from prophase to late metaphase: relationship to chromosome bands and their replication time

As chromosomes condense during early mitosis, their subbands fuse in a highly coordinated fashion. Subband fusion occurs when two large subbands flanking one minor subband come together to form one band, which takes on the cytological characteristics of the original flanking subbands. Using four different banding techniques--GTG (G-bands obtained with trypsin and Giemsa), GBG (G-bands obtained with BrdU and Giemsa), RHG (R-bands obtained by heating and Giemsa), and RBG (R-bands obtained with BrdU and Giemsa)--we studied subband fusion from prophase (1,250 bands per haploid set) to late metaphase (300 bands). To quantify the condensation process, a fusion index was established. We found that chromosomes contain preferential zones of condensation. From prophase to late metaphase, the early replicating subbands (R-subbands) fuse more readily with each other than do the late-replicating subbands (G-subbands). R-bands usually replicate early and condense late independently of the adjacent G-bands, which replicate late but condense early. Therefore, chromosome bands can undergo DNA replication and chromatin condensation relatively autonomously. Our data suggest that (1) chromosome replication and condensation are closely connected in time, (2) the metaphase bands represent independent units of chromatin condensation, and (3) the condensation process is an important feature of chromosome organization.     

Immunohistochemical localization of cyclic guanosine monophosphate (cGMP) on mouse fetal chromosomes

In previous immunohistochemistry studies, cyclic guanosine monophosphate (cGMP) has been found in polytene chromosomes of D. melanogaster, cGMP has not been found in mammalian metaphase chromosomes, but this could be due to loss of cGMP during staining. Thus the effect of different fixation techniques on the immunohistochemically detectable cGMP associated with metaphase chromosomes from mouse fetal tissue was examined. In chromosomes from cells fixed in 2% formalin, or unfixed cells dropped on slides preheated to 60 degrees C, there was diffuse cGMP staining. When cells were fixed in methanol:glacial acetic acid, 3:1, no chromosomal cGMP immunofluorescence was observed, whereas chromosomes from cells fixed in methanol:glacial acetic acid, 6:1, had different patterns of cGMP immunofluorescence depending on the temperature of the slides onto which the fixed cells were dropped. On slides prechilled to 4 degrees C, cGMP immunofluorescence outlined the chromosomes; on room temperature slides, faint chromosomal cGMP staining was observed, and on slides preheated to 68 degrees C or room temperature slides blown dry with hot air, the chromosomes had more intense diffuse cGMP immunofluorescence or distinct symmetrical bands of cGMP immunofluorescence. We have demonstrated the presence of cGMP in mammalian metaphase chromosomes. The different patterns of cGMP immunofluorescence observed may reflect variable preservation of chromosomal proteins that have binding sites for cGMP.     

Mapping G-bands on human prophase chromosomes.

OHNUKI's method for demonstrating coils in human metaphase chromosomes also reveals a fine G-band pattern on prophase chromosomes of sufficient clarity to justify an attempt at mapping. Maps are provided for each chromosome to show the maximum number of prophase bands observed, and an intermediate stage in chromosome contraction, tracing the pathways of apparent band fusion as the cell progresses to metaphase, is presented. The prophase bands on many chromosomes tend to occur in distinct groups, the members of which ultimately merge to give the dark G-bands of metaphase chromosomes. Every G-band of the standard metaphase chromosomes. Every G-band of the standard metaphase pattern is compounded from two or more prophase bands. In at least contracted prophase chromosomes examined, some bands are seen which have no obvious metaphase counterpart. There are marked similarities between banded prophases and the chromoomere pattern seen at meiotic prophase. However, since chromosome contraction is a dynamic process, agreement between maps will be expected only for corresponding degrees of chromosome contraction.     

Induction by chemical clastogens of aberrations in prematurely condensed interphase chromatin of Chinese hamster ovary cells

The clastogenic activities of diepoxybutane and bleomycin were comparatively studied on prematurely condensed interphase chromatin and metaphase chromosomes of Chinese hamster ovary cells. The yield of chromosomal aberrations was distinctly higher in G2-premature chromosome condensation as compared to metaphase. Most notably, the clastogenic activity of bleomycin was visible in premature chromosome condensation after application of much lower final concentrations than necessary for induction of chromosome aberrations in metaphase. In addition, the different mechanisms of action of both clastogens were reflected by the aberration yield in GI and G2 immediately after exposure. While bleomycin induced aberrations throughout all stages of interphase, diepoxybutane did not induce aberrations in GI or G2. Though certainly not a routine system for genotoxicity testing, premature chromosome condensation analyses provide a powerful opportunity to demonstrate relationships between DNA damage and repair, and the production of chromosomal changes at the site of their formation.Abbreviations BM bleomycin - BrdUrd bromodeoxyuridine - CHO Chinese hamster ovary - DEB diepoxybutane - DMSO dimethylsulfoxide - FCS fetal calf serum - PCC premature chromosome condensation, prematurely condensed chromosomes - PEG polyethylene glycol     

Complex relationships between 5-aza-dC induced DNA demethylation and chromosome compaction at mitosis

A variety of treatments with 5-azadeoxycytidine (5-aza-dC) were applied to cultured human lymphocytes during one to four cell cycles. The effect of 5-aza-dC on DNA methylation was studied by using an antibody against 5-methylcytosine on mitotic chromosomes. 5-Azadeoxycytidine is known to induce strong and permanent demethylation of DNA. Unexpectedly complex relationships were observed between DNA methylation status and chromatid/chromosome compaction. The most dramatic alteration of compaction at mitosis was observed when pre-replicative chromosomes had unifilarly demethylated DNA. The compaction of chromosomes was found to depend only partially on the methylation of their DNA at the time of mitosis. Our results suggest that alteration of DNA methylation prevents the synchronization of chromatin compaction, inducing premature (or delayed) chromosome condensation, and that a crucial step is the DNA methylation status of the pre-replicative chromosome.     

Chromosome bands and the subunit structure of Chinese hamster metaphase chromosomes

Isolated Chinese hamster chromosomes dissociate into a series of specific chromatin subunits approximately the size of stainable chromosome bands upon reduction of the divalent ion concentration during or after isolation. At high pH the chromatin in some bands is differentially removable during chromosome isolation, leaving a banded chromosome with a pattern typical of most G-band procedures. This provides an alternate molecular mechanism to explain the production of banded chromosomes by a variety of staining procedures. These results also suggest an approach to chromatin fractionation, using metaphase chromosomes as a starting material.     

The role of DNA replication in chromosome condensation

At metaphase, DNA in a human chromosome is estimated to be compacted at least 10,000 fold in length. However, the higher order mechanisms by which the chromosomes are organized in interphase and subsequently further condensed in mitosis have largely remained elusive. One generally overlooked participant in chromosome condensation is DNA replication. Many early studies of eukaryotic chromosome organization and cell fusions have suggested that DNA replication plays a role in chromosome compaction. Recent phenotypic analysis of Drosophila DNA replication mutants has revitalized this old idea. In this review, the role of DNA replication in chromosome condensation will be examined.     

Why plant chromosomes do not show G-bands

Summary Giemsa techniques have refused to reveal G-banding patterns in plant chromosomes. Whatever has been differentially stained so far in plant chromosomes by various techniques represents constitutive heterochromatin (redefined in this paper). Patterns of this type must not be confused with the G-banding patterns of higher vertebrates which reveal an additional chromosome segmentation beyond that due to constitutive heterochromatin. The absence of G-bands in plants is explained as follows: 1) Plant chromosomes in metaphase contain much more DNA than G-banding vertebrate chromosomes of comparable length. At such a high degree of contraction vertebrate chromosomes too would not show G-bands, simply for optical reasons. 2) The striking correspondence of pachytene chromomeres and mitotic G-bands in higher vertebrates suggests that pachytene chromomeres are G-band equivalents, and that this may also be the case in plants. G-banded vertebrate chromosomes are on the average only 2.3 times shorter in mitosis than in pachytene; the chromomeric pattern therefore still can be shown. In contrast, plant chromosomes are approximately 10 times shorter at mitotic metaphase; their pachytene-like arrangement of chromomeres is therefore no longer demonstrable.     

Proteome analysis of human metaphase chromosomes

DNA is packaged as chromatin in the interphase nucleus. During mitosis, chromatin fibers are highly condensed to form metaphase chromosomes, which ensure equal segregation of replicated chromosomal DNA into the daughter cells. Despite >1 century of research on metaphase chromosomes, information regarding the higher order structure of metaphase chromosomes is limited, and it is still not clear which proteins are involved in further folding of the chromatin fiber into metaphase chromosomes. To obtain a global view of the chromosomal proteins, we performed proteome analyses on three types of isolated human metaphase chromosomes. We first show the results from comparative proteome analyses of two types of isolated human metaphase chromosomes that have been frequently used in biochemical and morphological analyses. 209 proteins were quantitatively identified and classified into six groups on the basis of their known interphase localization. Furthermore, a list of 107 proteins was obtained from the proteome analyses of highly purified metaphase chromosomes, the majority of which are essential for chromosome structure and function. Based on the information obtained on these proteins and on their localizations during mitosis as assessed by immunostaining, we present a four-layer model of metaphase chromosomes. According to this model, the chromosomal proteins have been newly classified into each of four groups: chromosome coating proteins, chromosome peripheral proteins, chromosome structural proteins, and chromosome fibrous proteins. This analysis represents the first compositional view of human metaphase chromosomes and provides a protein framework for future research on this topic.