Morphological and cytofluorometric study of the giant cells of the trophoblast of the common vole]

The primary and secondary giant cells of trophoblast in placenta Microtus arvalis were studied. The giant polyploid nuclei are formed in result of series of successively proceeding endomitotic polyploidization of chromosomes. Two stages of endomitosis are described: endointerphase with the uniform net of thin chromatin threads and the stage when small round or rod-shaped paired chromosomes gather mostly under the nuclear membrane. Great number of round, oval, and complex-shaped nucleoli may be seen in nuclei during both stages of endomitosis, the number growing during polyploidization. The morphology of the chromosome-nucleolar apparatus involves peculiarities of the polyploidization mechanism in placenta Microtus arvalis trophoblast. Endomitosis occurs both in low and high-polyploid nuclei. Cytofluorometric determination of the DNA amount in nuclei polyploid nature. The degree of polyploidy of the trophoblast giant cells nuclei during terminal differentiation of placenta corresponds to 128c-512c, and some nuclei contain the DNA amount corresponding to 1024 and 2048 chromosomal sets. The cause of origin of the polyploid cells in trophoblast of rodents placenta is discussed.     

Comparison of homologous polytene chromosomes in Phaseolus coccineus embryo suspensor cells: morphological,autoradiographic and cytophotometric analyses

The functional behaviour of unpaired homologous polytene chromosomes (2n=22), was investigated in nuclei of Phaseolus coccineus embryo suspensor cells. Observations were carried out on the morphological level and after ³H-thymidine and ³H-uridine autoradiography. Histone and total protein contents in the chromatin were also investigated. It was shown that corresponding regions of homologous chromosomes may show different functional structures. ³H-thymidine incorporation demonstrated differences between homologues in both DNA synthesis leading to chromosome endoreduplication (polytenization) and DNA amplification (extra DNA synthesis). ³H-uridine autoradiography showed that homologous regions in a given chromosome pair may display three labeling patterns: i) both regions labeled; ii) both regions unlabeled; iii) one region labeled and the other unlabeled. These three states are found to occur in different cells of one and the same embryo suspensor. Differences between homologous chromosome regions were also found in the ratios between DNA and protein contents in their chromatin. These results, which show that the functional activity of homologous chromosomes of the same complement may greatly differ, are discussed in relation to the characteristics of the system investigated.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

Polyteny and endomitosis in supergiant trophoblast cells of the gray vole Microtus subarvalis

A cytomorphological study was made of peculiarly structured polytene chromosomes in supergiant trophoblast cells of Microtus subarvalis. The polyteny level was extremely high (over 1024C). The polytene chromosomes are characterized by a rather high degree of condensation of single chromosomes, and, as a consequence, close chromosome junctions and the typical disk pattern are lacking. The presence of complex nucleoli in the nuclei of these cells also testifies to a great detachment of chromonemes in polytene chromosomes of the studied supergiant trophoblast cells. Compared to other rodent species, a lower degree of chromoneme junction in the vole polytene chromosomes may cause their easy dissociation into single chromonemata, whose further condensation results in endomitotic chromosome formation. The chromosome depolytenization, earlier suggested from the analysis of interphase nucleus markers, has been traced here in detail. The process of polytene chromosome splitting was most obvious in the nucleolus-organizing chromosomes. A hony-combed nucleolus splits into numerous micronucleoli. The nucleus pattern becomes altered. Once in the polytene nucleus, chromosome bundles were located below the nuclear membrane and the central zone of the karyoplasm was not completely filled up. However, after dissociation of polytene chromosomes the whole karyoplasm was filled up with small nucleoli, and a thin layer of endomitotic chromosomes was seen beneath the nuclear membrane. The correlation between endomitosis and polyteny is discussed in terms of the dissociation of polytene chromosomes and formation of endomitotic chromosomes.     

Disproportionate numbers of rDNA loci in diploid and polyploid testis nuclei of gerris najas detected by fluorescence in situ hybridization

The replication state of rDNA in testes nuclei undergoing polyploidization by classical-type endomitosis was investigated in Gerris najas (Heteroptera) by means of fluorescence in situ hybridization. The number of just one rDNA locus per haploid genome was determined by in situ hybridization on meiotic nuclei. Additionally, DNA measurements of spermatids and testes nuclei were performed. Although regular duplication levels of nuclear DNA were found within the limits of the accuracy of the method, these did evidently not apply to the ribosomal genes. The comparison of the number of rDNA signals with the DNA content of 106 testis nuclei revealed drastic variations of the number of rDNA loci between individual nuclei with similar DNA content. Polyploid nuclei of the testis epithelium showed too low numbers of rDNA loci in relation to those expected from the levels of ploidy, while cyst cell nuclei displayed increased numbers of rDNA loci. The results indicate that the ribosomal genes are either underreplicated, or in part eliminated, during the endomitotic cycles of epithelium cell nuclei, but amplified in the cyst cell nuclei, probably already at their diploid stage.     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

Chromosome numbers, nuclear DNA content, and polyploidy in Consolea (Cactaceae), an endemic cactus of the Caribbean Islands

Polyploidy, an important mechanism of plant evolution, was investigated in Consolea, an endemic Caribbean opuntioid genus represented by nine subdioecious species with very narrow distributions, including species classified as rare or threatened. Standard chromosome counting and flow cytometric analyses were used to determine chromosome numbers and ploidy of each taxon. Compared to the base number (x = 11), the mitotic and meiotic counts indicated that there are seven hexaploid (2n = 66) and two octoploid species (2n = 88); no diploids were found. Histograms of intact nuclei confirmed that all species are polyploid, with C-DNA values ranging from 4.88-9.50 pg. The variation of DNA content was significantly higher for the octoploids than for the hexaploids. Male and female sexual morphs had similar DNA content, suggesting that there are no sex chromosomes. Cytomixis between cells and microsporocytes with no chromatin were observed. This provides a mechanism whereby gametes with variable chromosome numbers are produced, influencing reproduction and promoting speciation. In conclusion, C-DNA content and chromosome number separated Consolea species into two groups, which may correspond to two phylogenetic lineages or indicate that polyploidization occurred independently, with comparable effects on C-DNA content.     

Ploidy reduction and genome segregation in cultured carrot cell lines. I. Prophase chromosome reduction

Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner.The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.     

Studies on the Growth in Culture of Plant Cells: XVIII. NUCLEAR CYTOLOGY OF ACER PSEUDOPLATANUS SUSPENSION CULTURES

Established suspension culture strains of Acer pseudoplatanuswere analysed by chromosome counting and microdensitometricDNA measurements of individual nuclei. Comparison with the root-tipcomplement (2n=4x=52) showed the cultures to be entirely aneuploidwith modal chromosome numbers of approximately 75 and 135 plussome cells with 250–350. Analysis of the frequency distributionsof nuclear DNA values through the growth cycle of a batch cultureshowed that stationary phase cells accumulated in G1 of thecell cycle. Stationary phase DNA distributions could thus beused to indicate the chromosomal status of a culture withoutthe complication of G1 and G2 DNA values for each chromosomenumber. Root-tip and cultured cells showed a close correlationbetween DNA content and chromosome number indicating that structuralchanges and loss of chromosomes had been at random.     

Meiotic division and fusion of nuclei in multinucleate cells from the induced fusion of meiotic protoplasts of liliaceous plants

Multinucleate protoplasts were produced from meiotic cells at the zygotene and pachytene stages in a lily andTrillium, and their meiotic divisions were followed during subsequent culture. In each multinucleate, a complete synchrony of nuclear division was maintained throughout the meiotic process, and chromosome behavior appeared normal up to the metaphase stage. In most dinucleates, chromosome segregation movement was organized in a common spindle, and the daughter nuclei at the telophase appeared to envelope each other in the newly formed nuclear membrane. The cell was divided into two daughter cells by a common cell plate. Trinucleates were similarly converted to two cells with a hexaploid number of chromosomes. Some of the di- and trinucleates subsequently completed the second meiotic division with the formation of typical tetrad configurations. In giant cells with more than several nuclei, chromosomes separated at random but reaggregated into one giant resting nucleus, with no later cytokinesis. The rate of meiotic development in multinucleates was relatively slower in cells which contained greater numbers of nuclei.     

Chromosome evolution in fish: BrdU replication patterns demonstrate chromosome homeologies in two species of the genus Astyanax

A comparison of R-banding patterns obtained by 5-bromodeoxyuridine incorporation was made between the chromosomes of two fish species of the genus Astyanax (Characiformes: Tetragonopterinae), A. altiparanae with 2n = 50 chromosomes, and A. schubarti with 2n = 36 chromosomes. The two species present the highest and the lowest chromosome numbers found in this fish genus, respectively, for which the modal chromosome number is 50. R-band homeology was detected, involving eleven chromosomes of A. schubarti and seventeen chromosomes of A. altiparanae, indicating a close chromosomal relationship between the two species, in spite of their great difference in chromosome number. A chromosome fusion in the past history of the group was hypothesized as a possible cause of the discrepant chromosome numbers of the two species.     

Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae.

Naturally occurring strains of Candida albicans are opportunistic pathogens that lack a sexual cycle and that are usually diploids with eight pairs of chromosomes. C. albicans spontaneously gives rise to a high frequency of colonial morphology mutants with altered electrophoretic karyotypes, involving one or more of their chromosomes. However, the most frequent changes involve chromosome VIII, which contains the genes coding for ribosomal DNA (rDNA) units. We have used restriction fragment lengths to analyze the number and physical array of the rDNA units on chromosome VIII in four normal clinical strains and seven morphological mutants derived spontaneously from one of the clinical isolates. HindIII does not cleave the rDNA repeats and liberates the tandem rDNA cluster from each homolog of chromosome VIII as a single fragment, whereas the cleavage at a single site by NotI reveals the size of the single rDNA unit. All clinical strains and morphological mutants differed greatly in the number of rDNA units per cluster and per cell. The four clinical isolates differed additionally among themselves by the size of the single rDNA unit. For a total of 25 chromosome VIII homologs in a total of 11 strains considered, the variability of chromosome VIII was exclusively due to the length of rDNA clusters (or the number of rDNA units) in approximately 92% of the cases, whereas the others involved other rearrangements of chromosome VIII. Only slight variations in the number of rDNA units were observed among 10 random C. albicans subclones and 10 random Saccharomyces cerevisiae subclones grown for a prolonged time at 22 degrees C. However, when grown faster at optimal temperatures of 37 and 30 degrees C, respectively, both fungi accumulated higher numbers of rDNA units, suggesting that this condition is selected for in rapidly growing cells. The morphological mutants, in comparison with the C. albicans subclones, contained a markedly wider distribution of the number of rDNA units, suggesting that a distinct process may be involved in altering the number of rDNA units in these mutants.     

Timing of initiation of chromosome replication in individual Escherichia coli cells.

The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method. Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry. In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins. A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes. The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation. After inhibition of initiation and run-out of replication in rapidly growing B/r A and K-12 cultures, a small fraction of the cells (2-7%) contained 3, 5, 6 or 7 chromosomes. From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau). A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation. A similar pattern of chromosome number per cell was found after treatment of a recA strain.     

On the variation in chromosome number in Potamogeton pectinatus L.

To determine whether the large morphological differences that occur between populations of Potamogeton pectinatus L. have a genetic basis, the chromosome numbers of several populations were counted. Although several numbers were determined, no correlation between different numbers and populations could be established. The number was always close to 2n = 78, which is that most recorded for P. pectinatus in the literature. However, although some aberrant counts can probably be ascribed to incorrect interpretations of the preparations, some cells undoubtedly contain more than 78 chromosomes. Since the chromosomes of P. pectinatus are very small it is difficult to distinguish between “normal” and “B-chromosomes”, which may be the cause of the higher numbers.     

Molecular and morphological characterization of monosomic additions in Beta vulgaris,carrying extra chromosomes of B. procumbens or B. patellaris

DNA fingerprinting with three repetitive DNA sequences (OPX2, PB6-4 and Sat-121) was carried out on a set of 10 monosomic additions of Beta procumbens and 75 anonymous B. patellaris-derived monosomic additions in B. vulgaris, for characterization of the alien chromosomes at the DNA level. The probes are Procumbentes-specific and distributed over all chromosomes. Morphological characteristics were also used for the classification of B. patellaris monosomic addition families and for comparison with the morphology of the addition families of B. procumbens.DNA fingerprinting revealed unique patterns for almost all individual addition chromosomes of B. procumbens. However, it was concluded that chromosomes 1 and 6 of B. procumbens may be identical with the only difference that the chromosome referred to as 6 carries a susceptible allele for beet cyst nematode (BCN) resistance. In contrast, it was concluded that the two addition types with chromosome 2 are carrying different chromosomes of B. procumbens, so that one of them was renumbered to become the new chromosome 6.DNA fingerprinting of 75 anonymous B. patellaris-derived monosomic additions facilitated the identification and characterization of the alien chromosomes and the grouping of these additions into nine different groups. Several of these groups could be divided in two sub-groups on the basis of small differences in banding patterns. The results of the DNA fingerprinting led to the conclusion that B. patellaris most likely is an allotetraploid. It was also deduced that the BCN gene(s) in this species are homozygous and located on chromosome 1, while the pair of homoeologous chromosomes does not carry such BCN gene(s). Because of the allotetraploid nature of B. patellaris, preferential association occurs between the two homologous chromosomes containing the allele(s) for BCN resistance. Each group of B. patellaris addition families united by DNA fingerprinting had comparable morphological characteristics. Some of these morphological traits appeared to be chromosome-specific and were very useful for primary classification of the addition families. However, the present study showed that these morphological traits are not adequate for the identification of all alien chromosomes without the aid of additional markers. Because of similarities observed between molecular characteristics or the effects on plant morphology of several chromosomes of B. procumbens and B. patellaris it was concluded that B. procumbens could have been involved in the evolutionary history of B. patellaris.     

Meiosis and pollen mitosis in diploid and triploid Colocasia antiquorum Schott.

The relationship between diploid and triploid forms of Colocasia antiquorum Schott. was assessed through comparative meiotic and pollen mitotic studies. Owing to poor spreading of the chromosomes of both materials, karyological observations on pachytene nuclei were limited to a few chromosomes. Among the two nucleolar chromosomes and a metacentric, telochromomere-bearing chromosome of the diploid, the latter and one of the nucleolar chromosomes characterized by a heteropycnotic short arm were identified in both bivalent and trivalent associations in the triploid. The homologues in these cases were homomorphic and intimately paired. Two types of heteromorphic bivalents exhibiting partial pairing of homomorphic segments were also recorded in the triploid. Among the 14 bivalents of the diploid at diakinesis, two were nucleolus-associated. In the triploid, chromosomal associations at diakinesis included trivalents (2 to 9), bivalents and univalents, and the chiasma frequency per paired chromosome was lower than in the diploids. In 21.6 percent of the PMCs at this stage intragenomic pairing of one or two chromosomes was observed. Post-diakinesis stages in the diploid were regular while in the triploid they were marked by various irregularities in a majority of the cells. However, fertility (stainability), size and divisional frequency of pollen in both materials were remarkably similar. Chromosome numbers in pollen nuclei in the triploid ranged from 8 to 25. Based on these data an autopolyploid origin for the triploid Colocasia and a lower base number than the gametic chromosome number for this genus are advanced.     

Parental genome separation and elimination of cells and chromosomes revealed by AFLP and GISH analyses in a Brassica carinata x Orychophragmus violaceus cross

BACKGROUND AND AIMS: The phenomenon of parental genome separation during the mitotic divisions of hybrid cells was proposed to occur under genetic control in intergeneric hybrids between cultivated Brassica species and Orychophragmus violaceus (2n = 24). To elucidate further the cytological and molecular mechanisms behind parental genome separation, Brassica carinata (2n = 34) x O. violaceus hybrids were resynthesized and their chromosome/genomic complements analysed. METHODS: F(1) hybrids of the cross were obtained following embryo rescue, and were investigated for their cytological behaviour and subjected to genomic in situ hybridization (GISH) and amplified fragment length polymorphism (AFLP) to determine the contribution of parental genomes. KEY RESULTS: All the F(1) plants with high fertility closely resembled B. carinata in morphological attributes. These were mixoploids with 2n chromosome numbers ranging from 17 to 35; however, 34, the same number as in B. carinata, was the most frequent number of chromosomes in ovary and pollen mother cells (PMCs). GISH clearly identified 16 chromosomes of B. nigra in ovary cells and PMCs with 2n = 34 and 35. However, no O. violaceus chromosome was detected, indicating the presence of the intact B. carinata genome and elimination of the entire O. violaceus genome. However, some AFLP bands specific for O. violaceus and novel for the two parents were detected in the leaves. Cells with fewer than 34 chromosomes had lost some B. oleracea chromosomes. F(2) plants were predominantly like B. carinata, but some contained O. violaceus characters. CONCLUSIONS: The cytological mechanism for the results involves complete and partial genome separation at mitosis in embryos of F(1) plants followed by chromosome doubling, elimination of cells with O. violaceus chromosomes and some introgression of O. violaceus genetic information.     

Preliminary karyotype and chromosomal localization of ribosomal DNA sites in white spruce using fluorescence in situ hybridization.

We have localized the major ribosomal DNA (rDNA) loci on metaphase chromosomes and in interphase nuclei of white spruce (2n = 24) by fluorescence in situ hybridization. Hybridization sites of the biotin-labelled rDNA probe were detected using antibody-fluorochrome conjugates and a confocal laser scanning microscope. White spruce has at least 12, and possibly as many as 14, rDNA sites, 1 site present on each of seven separate chromosome pairs. This is one of the highest numbers of rDNA loci yet reported among plant species. The position of the rDNA loci together with secondary constriction patterns permit, for the first time, all homologous pairs of white spruce chromosomes to be distinguished. We discuss the application of molecular cytogenetics in studies relating to the organization and evolution of DNA sequences within conifer genomes.