Isolation,fractionation and biochemical analysis of the metaphase chromosomes of Microtus agrestis

A method has been developed for isolating metaphase chromosomes from Microtus agrestis fibroblasts in relatively large quantities with recovery of about 50% of the chromosomes present in the metaphase cells. The method employs pressure homogenisation to release the chromosomes from the cells. The average chemical composition of the Microtus chromosome preparations is 24.6% DNA, 19.9% RNA and 55.5% protein. The isolated chromosomes were fractionated by sedimentation velocity in a density gradient into three size groups in one of which 75–80% of the chromosomes were the large sex-chromosomes. The relative composition of this fraction containing most of the heterochromatin of the cell was DNA: 100, RNA: 59, acid-soluble protein: 54, acid-insoluble protein: 178. — Disc electrophoresis studies revealed no significant difference in the histone patterns between the euchromatic and heterochromatic chromosomes of the three chromosome size-groups. Metaphase chromosomes appear to have a lower lysine-rich histone content than interphase nuclei.     

Pseudomultiple production in Ageneotettix deorum deorum

Meiosis in Ageneotettix deorum deorum is characterised by extensive pseudomultiple formation during prophase. The association of non-homologous chromosomes takes place prior to pairing and chiasma formation and occurs to a varying degree in all prophase cells. These pseudomultiples originate during interphase as a consequence of the association of heterochromatin. All autosomes carry procentric heterochromatic segments of variable size and the L₁, L₃ and M₅ chromosomes also possess terminal heterochromatic regions. The association of non-homologous chromosomes during zygotene and pachytene does not appear to impede pairing or the frequency and distribution of chiasmata. — A majority of the pseudomultiples dissociate after diakinesis, during orientation and congression on the spindle. However in 4% of the cells examined, associations, mainly quadrivalents, persist through metaphase. — Heterochromatic associations of non-homologous chromosomes are again evident during second prophase, though here they involve only the centric heterochromatic regions; 9% of these associations persist through second metaphase. — The nature and behaviour of the pseudomultiples in Ageneotettix are pertinent to the interpretation of terminal associations in other Orthoptera and provide evidence that persistent associations can arise following a non-chiasmate association.On educational leave from the Forest Research Laboratory, Fredericton, N.B. Canada.     

Absence of the nucleolar constriction in the division of the generative nucleus of Haplopappus gracilis

The nucleolar constriction which is a structural characteristic of the chromosomes of Haplopappus gracilis (n = 2) is not formed in the division of the generative nucleus in pollen grains. Some brief discussions are given on its absence.     

Chromosome distribution in a23 chinese hamster fibroblasts

This study deals with a systematic chromosome position analysis of 116 anti-mitotic and hypotonic treated a₂₃ chinese hamster cells. No chromosome or pair of chromosomes was found to be located nearer the center or the periphery of the metaphase plate than would be expected by the reference distribution. The homologous chromosomes of pair 2 lie nearer to each other but they do not form a specific angle. The same relative position was shown for the chromosome groups 1–2, 1-E1 and 2-E5 (E standing for an extra chromosome). On the other hand the chromosomes of the combinations X-7, X-8, 7–8, 8–11 and X-E2 were lying further from each other, while chromosomes 10-E1 had a greater mean angle. The non random distribution of the chromosomes 1 and 2 may be interpreted as function of their possibly more frequent participation in the organization of nucleoli. — To obtain more information about the influence of preparation techniques on the alteration of the chromosome position in metaphase plates, this study deals with some overall considerations about chromosome position. It is shown that in a₂₃ cells the smaller chromosomes do not tend to lie nearer the metaphase plate center (as it happens in human cells). Also a significant correlation between the chromosome position with respect to the metaphase plate center and the mean interchromosomal distances was not found in this type of cells.     

Relative order determination of four Yp cosmids on metaphase and interphase chromosomes by two-color competitive in situ hybridization

Summary Two-color competitive in situ hybridization was used to cytogenetically order four Yp cosmid probes, located in the pseudo-autosomal and TDF regions. The probes were hybridized by pairs to metaphase and interphase chromosomes. On metaphase chromosomes, determination of order between sequences separated by 3 Mb from each other was possible on a statistical basis, whereas the relative position of sequences 0.6Mb apart could not be determined. On interphase chromosomes the complete order between sequences separated by 0.6– 6Mb was obtained rapidly by measuring the distances between two cosmid spots of every cosmid pair used in 28 to 60 nuclei. Results demonstrate the potential power of fluorescent in situ hybridization at interphase for high resolution cosmid mapping.     

Meiosis in Drosophila melanogaster

Individual bivalents or chromosomes have been identified in Drosophila melanogaster spermatocytes at metaphase I, anaphase I, metaphase II and anaphase II in electron micrographs of serial sections. Identification was based on a combination of chromosome volume analysis, bivalent topology, and kinetochore position. — Kinetochore microtubule numbers have been obtained for the identified chromosomes at all four meiotic stages. Average numbers in D. melanogaster are relatively low compared to reported numbers of other higher eukaryotes. There are no differences in kinetochore microtubule numbers within a stage despite a large (approximately tenfold) difference in chromosome volume between the largest and the smallest chromosome. A comparison between the two meiotic metaphases (metaphase I and metaphase II) reveals that metaphase I kinetochores possess twice as many microtubules as metaphase II kinetochores. — Other microtubules in addition to those that end on or penetrate the kinetochore are found in the vicinity of the kinetochore. These microtubules penetrate the chromosome rather than the kinetochore proper and are more numerous at metaphase I than at the other division stages.     

A structural anomaly of the satellite in Haplopappus gracilis

Summary A cytological examination of a population of Haplopappus gracilis revealed one plant possessing a heteromorphic satellited pair of chromosomes in all the root tip cells. The cytology and the detailed morphology of this anomaly are described.     

Nucleolar behaviour in Triticum

The maximum number of major nucleoli (macronucleoli) per nucleus of hexaploid, tetraploid and diploid wheat, Aegilops speltoides and Ae. squarrosa corresponded to the number of satellited chromosomes of each species. Smaller nucleoli (micronucleoli) were rare or absent in all of these species except the hexaploid, in which they were predominantly organized on chromosome arm 5D^s. — Fewer than the maximum number of macronucleoli in a mitotic interphase nucleus resulted from fusion of developing nucleoli. Enforced proximity of nucleolus-organizing regions resulted in more frequent fusion of nucleoli. — Analyses of related interphase nuclei showed that nucleoli, and hence probably chromosomes, undergo limited movement during mitotic interphase. These observations also indicate that specific chromosomes do not occupy specific sites in the interphase nucleus.     

Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes

The chromatin in interphase nuclei is much less condensed than are metaphase chromosomes, making the resolving power of fluorescence in situ hybridization (FISH) two orders of magnitude higher in interphase nuclei than on metaphase chromosomes. In mammalian species it has been demonstrated that within a certain range the interphase distance between two FISH sites can be used to estimate the linear DNA distance between the two probes. The intephase mapping strategy has never been applied in plant species, mainly because of the low sensitivity of the FISH technique on plant chromosomes. Using a CCD (charge-coupled device) camera system, we demonstrate that DNA probes in the 4 to 8 kb range can be detected on both metaphase and interphase chromosomes in maize. DNA probes pA1-Lc and pSh2.5·SstISalI, which contain the maize locia1 andsh2, respectively, and are separated by 140 kb, completely overlapped on metaphase chromosomes. However, when the two probes were mapped in interphase nuclei, the FISH signals were well separated from each other in 86% of the FISH sites analyzed. The average interphase distance between the two probes was 0.50 µm. This result suggests that the resolving power of interphase FISH mapping in plant species can be as little as 100 kb. We also mapped the interphase locations of another pair of probes, ksu3/4 and ksu16, which span theRp1 complex controlling rust resistance of maize. Probes ksu3/4 and ksu16 were mapped genetically approximately 4 cM apart and their FISH signals were also overlapped on metaphase chromosomes. These two probes were separated by an average of 2.32 µm in interphase nuclei. The possibility of estimating the linear DNA distance between ksu3/4 and ksu16 is discussed.     

Evidence against somatic association in hexaploid wheat

In mitotic interphase nuclei, nucleolus-organizing regions of homologous chromosomes lay no closer to each other than nucleolus-organizing regions of non-homologous chromosomes. — Mitotic metaphase studies of distances between satellites and also between telocentrics showed no greater nearness of homologues than of non-homologues. — These studies fail to support the concept of somatic association in hexaploid wheat.     

Nucleoli and ribosomal RNA cistron numbers in Hordeum species and interspecific hybrids exhibiting suppression of secondary constriction

The interspecific hybrids of Hordeum exhibit selective suppression of secondary constriction formation in the chromosome(s) contributed by one of the two parents. A comparison of the number of SAT (secondary constriction) chromosomes in the metaphase cells and the maximum number of nucleoli in interphase cells revealed that the chromosomes capable of organising nucleoli were not always reflected through secondary constriction formation. — The rDNA (DNA complementary to rRNA) amounts were estimated by DNA-rRNA filter hybridisation in diploid and polyploid species of Hordeum and their hybrids. While similar rDNA proportions were present in diploid and autotetraploid lines of H. bulbosum, there were up to threefold differences between H. vulgare and allohexaploids. Furthermore, differences were also apparent between species of same ploidy level. Ribosomal RNA (18S+5.8S+26S) cistron numbers in each of the five experimental hybrids exhibiting the selective suppression of secondary constriction formation revealed no selective loss of rDNA. — The presence of a higher number of nucleoli than the number of SAT chromosomes seen and the presence of expected number of rRNA cistrons suggest that the suppression of secondary constriction formation is not due to selective loss of rDNA.     

Nucleolar organizer activity in wheat,rye and derivatives analyzed by a silver-staining procedure

Nucleolar activity was analyzed in wheat (Triticum sp.), rye (Secale cereale) and several types of wheat-rye derivatives using a modified, highly reproducible, silver staining procedure (Lacadena et al. 1984). A comparative analysis of the nucleolar organizer regions (NORs) of somatic metaphase chromosomes was made by phase contrast, C-banding, and silver staining. The frequency distribution of the number of nucleoli visualized at interphase by silver staining was also used to infer the activity of NORs. The results agree quite well with data from in situ hybridization reported by other authors. The behavior of euploid, ditelosomic and nulli-tetrasomic plants of common wheat showed the relative nucleolar activity of the four organizer chromosomes to be: 6B > 1B > 5D > 1A. — Several types of wheat-rye derivatives were analyzed: interspecific hybrid, triticale, addition and substitution lines, and plants with the genome constitutions, AABBDR, ABDR + 5D, ABRR, and ABRRR. In all cases the nucleolar organizer chromosome 1R of rye was suppressed by the presence of wheat chromosomes.     

Flow-cytometric characterization and sorting of plant chromosomes

Summary Flow cytometric measurements of DNA frequency distribution were used to follow the synchronization process in suspension cells from Haplopappus gracilis (2n=4). Metaphase chromosomes were isolated from these synchronized cells and both the acro- and metacentric chromosomes were sorted by flow cytometry based on the different DNA contents. Possible applications of this procedure in fundamental genetics as well as practical plant breeding are discussed.     

An effect of exogenous thymidine on the cell cycle in Haplopappus gracilis

The study of an effect of exogenous thymidine on the mitotic cycle demonstrated that a 30 minute exposure to unlabeled and to tritiated thymidine at a concentration of 2.9 × 10^?6 M was sufficient to cause a significant increase in the mitotic index of root meristem cells of Haplopappus gracilis. An analysis of the data revealed that this was due to the prolongation of metaphase rather than to an increase in the actual number of cells entering division.     

Chromosome distribution and catenation in Rhoeo spathacea var. concolor

The twelve chromosomes of Rhoeo spathacea variety concolor are arranged in a definite sequence in a ring at meiosis. Identification of all the 12 chromosomes was possible in 119 diakinesis and metaphase I cells. — Pollen viability was measured to be 36.54% by cotton blue staining procedure. Forty five of 56 metaphase I cells (80.36%) had adjacent distribution. Each of the 12 chromosomes was equally likely to be involved in adjacent distribution regardless of their sizes and heterobrachialness. Adjacent distribution occurred randomly at each arm-position in the ring regardless of the lengths of the arm-pairs. — The most frequent chromosome configuration at diakinesis and metaphase I was a chain-of-12 chromosomes (41.18%). Cells with 1 to 4 chains of chromosomes were observed. The observed frequencies of various configurations were in good agreement with the calculated frequencies. The mean number of chiasmata was 10.90 per cell and 0.908 per pair of chromosome arms. The 131 chiasma failures were distributed at random among the 12 arm-positions. Since the lengths of arm-pairs in the ring vary, the randomness may mean that chiasma formation was limited to short terminal segments on all chromosomes.     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.     

The discriminating fluorescence patterns of the chromosomes of Drosophila melanogaster

Mitotic and salivary gland chromosomes of D. melanogaster show striking fluorescent patterns when stained with Quinacrine. In the salivary gland chromosomes there are up to five strongly fluorescing bands located on the fourth chromosome and at the proximal end of the X chromosome.—In mitotic cells the Y chromosome shows four fluorescent segments and other fluorescent regions are found proximally on the third pair and on the X chromosome. It is, therefore, possible to distinguish male and female interphase cells by their patterns of fluorescence.—A comparison between the position of heterochromatic, late replicating and fluorescing segments in the mitotic chromosomes, shows differences which demonstrate, for the first time, the chemical, morphological and genetical diversity of these three types of segments.     

Arrangement and association of somatic chromosomes induced by chloramphenicol in barley

Chromosome number in the cells of the first division cycle in the root tip of Hordeum vulgare (2n=14) was apparently reduced from 2n to n by the chloramphenicol (CAP) treatments in early S, S late and/or early G ₂ stages. Morphological observations and measurements of relative DNA content per cell indicated that such reduction was due to tight alignment of chromosomes in pairs. —It was supposed that homologous chromosomes are close together in the interphase nucleus, and their successive association up to mitotic prometaphase was induced by the CAP treatment.     

Genetic processes in intergeneric cell hybrids Atropa + Nicotiana

Summary The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120–150 chromosomes including 50–70 of Atropa and 50–90 of Nicotiana. Other clones are polyploid and possess 200–250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.Dedicated to Professor G. Melchers with gratitude