Natural variation in nuclear characters of meristems in Vicia faba

The natural variation in chromosome size in root-tip and shoot apex meristems of Vicia faba has been studied. Chromosomes in main root-tips of one week old plants are 2–3 times larger than those in small lateral root-tips of 3 week old plants. While the nuclear DNA content remains constant, the nuclear RNA and nuclear histone contents show a positive linear correlation with chromosome volume. The DNA: histone ratio varies and is lowest in cells with large chromosomes. Nuclear volume and chromosome volume are not linearly related and changes in nuclear density therefore seem likely. A positive linear relationship between chromosome volume and mitotic index in colchicined squashes is demonstrated. The results strongly suggest that variation in chromosome size indicates a corresponding change in the rate of cell metabolism and may well reflect change in genetic activity.     

AN ANALYSIS OF HETEROCHROMATIN IN MAIZE ROOT TIPS

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15–20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.     

Localization of satellite DNAs in the chromosomes of the guinea pig

The in situ hybridization method has been used to investigate the localization of each of the three satellite DNAs present in the genome of the guinea pig. Purified fractions of the satellite DNAs were utilized as templates for synthesis of ³H-labeled complementary RNA (cRNA) by E. coli RNA polymerase, then each cRNA was hybridized to metaphase spreads of embryonic guinea pig cells. The cRNAs of all three satellite DNAs hybridized predominantly to the centromeric region of the chromosomes. The cRNAs of satellite DNAs II and III hybridized to all chromosomes except the Y chromosome. The cRNA of satellite DNA I did not hybridize to the Y chromosome nor to two pairs of small acrocentric chromosomes. Satellite II cRNA hybridized to the telomeric region of chromosomes 3 and 4.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Delayed termination of nuclear histone doubling after premeiotic DNA synthesis inTriturus vulgaris male meiosis

It has been shown by means of double wavelength cytophotometry of DNA (Feulgen reaction) and histone (fast green, pH 8.2) inTriturus vulgaris spermatocytes that the doubling of DNA content in nuclei terminates at the end of preleptotene to beginning of leptotene whereas the doubling of histone content begun at premeiotic interphase is delayed and proceeds till the end of leptotene to beginning of zygotene. As a result preleptotene spermatocytes contain approximately 4C DNA and only 3C histone. Histone content in leptotene amounts to 93% of 4C, and in zygotene, pachytene and metaphase I both DNA and histone contents equal 4C. Thus, the temporal pattern of nucleo-histone doubling in meiotic chromosomes ofT. vulgaris differs from the synchronous DNA and histone doubling in mitotic chromosomes of all previously studied species. The delay of histone doubling inT. vulgaris meiocytes is less pronounced than in the previously studied insectsAcheta domestica andPyrrhocoris apterus where the histone content amounts to 3C in leptotene—zygotene and the equal histone/DNA ratio is restored only in pachytene.—Responsibilities for this phenomenon and its biolgoical sinnificance are discussed in connection with recent hypotheses concerning mechanisms of homologous chromosome pairing.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

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.     

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     

Immunofluorescent studies of human chromosomes with antibodies against phosphorylated H1 histone

One of the prominent cell cycle-related modifications of histone proteins whose function remains unresolved is the phosphorylation of linker histone H1. In this work we have used indirect immunofluorescence on human cells with antibodies that are specific for phosphorylated histone H1 to examine the cellular distribution and chromosome association patterns of this protein. With confocal microscopy on whole cells, strong immunofluorescence was seen in association with mitotic chromosomes as well as a prominent punctate pattern of labeling throughout the mitotic cell, whereas interphase cells showed very little, if any, specific fluorescence. Multiple patterns of fluorescence distribution were detected with metaphase chromosomes, ranging from apparent tight colocalization with the DNA to expanded ”puffy” mitotic figures to an amorphous network of staining. It was also shown that the ability to label chromosomes could vary drastically with different fixation procedures, adding further complications to interpretation of the potentially complex role of phosphorylated histone H1 in chromatin condensation or decondensation. Received: 8 September 1999; in revised form: 14 September 1999 / Accepted: 17 September 1999     

Cytogenetic analysis of metaphase chromosomes from pupal testes of four mosquito species using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization technique (FISH)

The DNA probes, P1887, P2405, P2056 (being specific tags for Aedes aegypti genes coding for ribosomal RNA) and a centric heterochromatin probe, K20-1A5, were chosen to hybridize the metaphase chromosomes from the testes of four mosquito species, Culex pipiens, Aedes aegypti, Aedes albopictus and Aedes triseriatus. In addition, a single plasmid, P2392, which contained the three probes, P1887, P2405 and P2056, was also used as chromosome landmark in aedine species. Only the Aedes aegypti metaphase chromosome 1-specific tag, P1887, was conserved in Aedes albopictus, Aedes triseriatus, and Culex pipiens metaphase chromosomes. Aedes triseriatus exhibited two gene loci, on chromosomes 1 and 3, coding for ribosomal RNA per haploid genome. When the specific probes for chromosomes 2 and 3, 2405 and 2056, were used in the fluorescence in situ hybridization technique against the metaphase chromosomes the fluorescent signals were not seen in Aedes albopictus, Aedes triseriatus or Culex pipiens. Also the centric heterochromatin probe, K20-1A5, exhibited strong fluorescent signals on chromosomes 1, 2 and 3 of Aedes aegypti. These fluorescent signals were not observed in metaphase chromosomes derived from the other aedine species, indicating that the centromere sequence can vary within the species.This paper was presented at the Second Arab Conference on Biotechnology and Genetic Engineering, held in the Kingdom of Bahrain, 15–17 April, 2002 and is published here with the endorsement of the Co-ordinator of the Scientific Committee, Professor Essam H. Ghanem, University of Bahrain. Its publication has been delayed because of the ill health of the senior author. Other papers from this conference were published in the July 2003 issue (vol. 19, no. 5).     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination‐poor region comprises more than half of barley chromosome 3H

Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single‐copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination‐poor region is preferentially marked by a heterochromatin‐typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin‐typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape.     

Cytological investigation of Triticale

Summary A cytological investigation of 15 different 56-chromosome Triticale and 16 Triticale with 42 chromosomes was carried out. 4 were primary Triticale and 12 were secondary Triticale. Chromosome pairing was not disturbed; 21 and 28 bivalents were found in the hexaploids and octoploids, respectively. Meiotic irregularities were established, however, in all the Triticale studied; in octoploids the frequency of the irregularities was 22–88% and in hexaploids it was 12–87%.In metaphase and anaphase asynchronous separation of chromosomes was noted. Incompatibility between wheat and rye genomes and the inactivation of single loci of rye chromosomes are suggested as the main causes of the irregularities in meiosis.Mitotic disturbances were found in all the amphidiploids. The frequency of anomalies in mitosis was considerably lower than in meiosis: in octoploids they made up 5%–11% and in hexaploids 6.2%–15.2%. In all the amphidiploids studied chimera plants were found containing pollen mother cells with different chromosome numbers. The chromosome number in the aneuploid cells varied from 8–48 in hexaploids and from 8–62 in octoploids. Octoploid Triticale had 29.4%–72.9% aneuploid pollen mother cells, while hexaploid Triticale had 5-2%–55-7%.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> root induction in weedy <Emphasis Type="Italic">amaranthus</Emphasis> species to obtain mitotic chromosomes

Summary Mitotic chromosome analysis has proven to be an important tool in monitoring the potential for genetic exchange among related plant species. One major obstacle to using mitotic chromosome analysis in any species is obtaining large numbers of clear, well-spread metaphase chromosomes necessary to perform cytological techniques such as chromosome banding and fluorescent in situ hybridization. The ability to obtain good chromosome spreads is in part determined by the number and morphology of the roots, which contain the metaphase tissue. Many Amaranthus species produce very thin, delicate roots. The technique used in the process described herein provides for much more substantial roots, allowing for higher probability of obtaining well-spread metaphase chromosomes. Seeds were planted in a soilless mixture, and then cuttings and leaves were taken from the plants. The cuttings were sterilized and placed in Murashige and Skoog (MS) media, while leaf tissue was analyzed by flow cytometry, both pre-and post-propagation, to obtain DNA contents. No changes in DNA content were observed. The in vitro procedure produced significantly larger roots than were produced in soilless mix. Furthermore, all of the in vitro roots observed had 32 chromosomes of normal morphology. In vitro root propagation allowed large numbers of roots to be obtained from a single plant, thereby resulting in increased probability of obtaining cells with metaphase chromosomes that reflected the original plants' chromosome numbers and therefore may be used for molecular cytogenetic analysis.     

Reverse fluorescent chromosome banding with chromomycin and DAPI

Two DNA binding guanine-specific antibiotics, chromomycin A₃ (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.     

Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with ³H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S+18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S+18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. — In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.