Maize centromere mapping: a comparison of physical and genetic strategies

Centromere positions on 7 maize chromosomes were compared on the basis of data from 4 to 6 mapping techniques per chromosome. Centromere positions were first located relative to molecular markers by means of radiation hybrid lines and centric fission lines recovered from oat-maize chromosome addition lines. These centromere positions were then compared with new data from centric fission lines recovered from maize plants, half-tetrad mapping, and fluorescence in situ hybridizations and to data from earlier studies. Surprisingly, the choice of mapping technique was not the critical determining factor. Instead, on 4 chromosomes, results from all techniques were consistent with a single centromere position. On chromosomes 1, 3, and 6, centromere positions were not consistent even in studies using the same technique. The conflicting centromere map positions on chromosomes 1, 3, and 6 could be explained by pericentric inversions or alternative centromere positions on these chromosomes.     

Evidence for a relatively random array of human chromosomes on the mitotic ring

We used fluorescence in situ hybridization (FISH) to study the positions of human chromosomes on the mitotic rings of cultured human lymphocytes, MRC-5 fibroblasts, and CCD-34Lu fibroblasts. The homologous chromosomes of all three cell types had relatively random positions with respect to each other on the mitotic rings of prometaphase rosettes and anaphase cells. Also, the positions of the X and Y chromosomes, colocalized with the somatic homologues in male cells, were highly variable from one mitotic ring to another. Although random chromosomal positions were found in different pairs of CCD-34Lu and MRC-5 late-anaphases, the separations between the same homologous chromosomes in paired late-anaphase and telophase chromosomal masses were highly correlated. Thus, although some loose spatial associations of chromosomes secondary to interphase positioning may exist on the mitotic rings of some cells, a fixed order of human chromosomes and/or a rigorous separation of homologous chromosomes on the mitotic ring are not necessary for normal mitosis. Furthermore, the relative chromosomal positions on each individual metaphase plate are most likely carried through anaphase into telophase.     

Quantitative studies on the arrangement of human metaphase chromosomes. IX. Arrangement of chromosomes with and without spindle apparatus

Summary The spatial relationships in human male metaphase cells treated with and without colcemide were compared with each other. The following results were obtained: (1) In normal male metaphases the overall distributions of chromosomal distances regardless of chromosome identification numbers did not show normal distribution, neither in the colcemid-free sample nor in the colcemide-treated sample. (2) In both samples larger chromosomes showed a more peripheral position, and smaller chromosomes showed a more central position. This finding was statistically significant. (3) No differences between the two samples could be observed concerning the following parameters: overall distributions of the centromere-centromere distances, distributions of the distances between the homologous chromosomes (except the small acrocentric chromosomes), rank positions of the mean distances between homologous chromosomes, and rank positions of the mean distances of the different chromosomes from the center of the mitosis (except few chromosomes). (4) Visible, but not statistically accessible, differences appeared between the two samples in respect to rank positions of the mean distances of all possible acrocentric pairing groups, rank positions of the mean distances of the homologous acrocentric chromosomes from the center of the mitosis, and distances of the X chromosome from the center of the mitosis. (5) Statistically significant differences appeared between the two samples with respect to distance distributions of the small acrocentric chromosomes and positions of the chromosomes 1, 16, 18, Y, and 21, 22 in relation to the center of the mitosis.     

Changes in chromosome positioning may contribute to the development of diseases related to X-chromosome aneuploidy

The radial positions of the centromeric regions of chromosomes 1 and X were determined in normal male fibroblasts (XY) and in fibroblasts from a patient with a rare case of XXXXY polysomy. The centromeric regions and presumably the whole territories of active X chromosomes were demonstrated to occupy similar, although not identical, positions in XY and XXXXY cells. The centromeres of inactive X chromosomes (Barr bodies) were located closer to the nuclear periphery as compared with the centromeres of active X chromosomes. In addition, it was established that the nuclear radial position of gene-rich chromosome 1 was changed in XXXXY cells as compared to normal XY cells. The data are discussed in the context of the hypothesis postulating that changes in nuclear positioning of chromosomal territories induced by the presence of extra copies of individual chromosomes may contribute to the development of diseases related to different polysomies.     

Specific radial positions of centromeres of human chromosomes X, 1, and 19 remain unchanged in chromatin-depleted nuclei of primary human fibroblasts: evidence for the organizing role of the nuclear matrix

Radial positions of centromeres of human chromosomes X, 1, and 19 were determined in the nuclei of primary fibroblasts before and after removal of 60%-80% of chromatin. It has been demonstrated that the specific radial positions of these centromeres (more central for the chromosome 19 centromere and more peripheral for the centromeres of chromosomes 1 and X) remain unchanged in chromatin-depleted nuclei. Additional digestion of nuclear RNA did not influence this specific distribution. These results strongly suggest that the characteristic organization of interphase chromosomes is supported by the proteinous nuclear matrix and is not maintained by simple repulsing of negatively charged chromosomes.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

Karyotype analysis of four Vicia species using in situ hybridization with repetitive sequences

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S-25S and 5S rDNA, telomeres) and genus-specific satellite repeats (VicTR-A and VicTR-B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR-A and -B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied.     

Study of the organization of polytene chromosomes in the nuclei of Drosophila melanogaster pseudonurse cells with the use of the DNA library of the Drosophila orena chromocenter

The location of the Drosophila orena chromocenter in polytene chromosomes of pseudonurse cells of the D. melanogaster ovaries (the otu11 mutation) and salivary glands has been studied. Numerous sites of location of the D. orena chromocenter DNA have been found throughout the length of D. melanogaster chromosomes. The specific distribution of the binding sites for the DNA probe has made it possible to identify chromosomes and analyze their mutual positions in the three-dimensional space of the nuclei of pseudonurse cells. The mutual positions of chromosomes have been found to vary, the pericentromeric regions of different chromosomes differing from one another in associative ratios.     

RING-POSITION AND FREQUENCY OF ADJACENT DISTRIBUTION OF MEIOTIC CHROMOSOMES IN RHOEO SPATHACEA

The morphological sequence of the twelve chromosomes around the ring as worked out by Sax is reaffirmed with slight corrections of the centromere position on three chromosomes: Aa, fA, and Dd. Adjacent distribution was found in 53/120 MI PMC (44.2%). Ring-position analysis was achieved in 34 of the 53. There were 127 chromosomes and 66 arm-pairs involved in adjacent distribution in these 34 MI PMC. Adjacent distributions occurred at random among the twelve chromosome positions and among the twelve arm-pair positions. There were eleven instances among the 66 arm-pairs (16.7%) of adjacent distribution despite free ends due to chiasma failure. Up to four consecutive chromosomes may pass to the same pole. Not all cells with 6–6 distribution are genetically balanced. Distribution of 7–5 occurred in 24/120 AI PMC (20.0%). Another nine (7.5%) in the same sample had one or more lagging chromosomes. At MI, three PMC had 8–4 distribution, but none such were seen at AI.     

Bivariate flow karyotyping of human chromosomes: evaluation of variation in Hoechst 33258 fluorescence, chromomycin A3 fluorescence, and relative chromosomal DNA content.

The total variation of chromosome peak positions, in bivariate distributions of Hoechst 33258 and chromomycin A3 fluorescence of 19 healthy individuals, was compared with the experimental variation, determined from 23 bivariate distributions of chromosomes prepared separately from a single cell lineage. The experimental variation in Hoechst and chromomycin fluorescence and the relative chromosomal DNA content were determined from experiments performed over several days. The additional variance contributed by time was the same as the daily variance. The accuracy by which the relative chromosomal DNA content can be calculated from bivariate peak positions was investigated. A least squares method was used to fit the distributions of relative DNA content, obtained, respectively, from mono- and bivariate flow analyses of chromosomes from the same cell lineage. In general the DNA contents match quite well, but for a few chromosomes a difference was found, statistically discernible at the 5% level. The average relative chromosomal DNA content of the chromosomes from the 19 normal individuals, calculated from bivariate peak positions, showed a linear relation with the estimates published by other investigators.     

Chromosomes participating in translocations typical of malignant hemoblastoses are also involved in exchange aberrations induced by fast neutrons

Repeated triple-color fluorescence in situ hybridization was used for the detection of exchange aberrations among 10 selected chromosomes of human lymphocytes irradiated with three doses of fast neutrons with a mean energy of 7 MeV. In each hybridization two different pairs of chromosomes were stained. Defined stage positions of metaphases on a slide were stored on a hard disk and an automatic scan of images according to these positions was performed after six successive hybridizations. In this way we obtained six different images of the same metaphase with differently stained pairs of chromosomes and centromeres. The comparison of these images enabled the identification of mutual exchanges between chromosomes 1, 2, 3, 4, 8, 9, 12, 14, 18 and 22. The frequencies of exchanges were not linearly proportional to the molecular weight of interacting chromosomes. The most significant were exchanges between chromosomes 14/18, 14/8, 18/8, 8/3, 1/14, 1/8, 3/18, 3/14 and 9/22. The results indicate significant interactions between chromosomes involved in translocations in B-cell non-Hodgkin's lymphoma and chronic myeloid leukemia. We propose that the reason for the high frequency of exchanges between these chromosomes is their proximity in the cell nucleus. It may also be one of the reasons for the induction of specific translocations leading to malignant transformation of cells.     

Localization of translocation breakpoints in somatic metaphase chromosomes of barley

Karyotype analyses based on staining by acetocarmine followed by Giemsa N-banding of somatic metaphase chromosomes of Hordeum vulgare L. were carried out on 61 reciprocal translocations induced by X-irradiation. By means of computer-based karyotype analyses all of the 122 breakpoints could be localized to defined sites or segments distributed over the seven barley chromosomes. The pre-definition of translocations with respect to their rearranged chromosome arms from other studies rendered it possible to define the break positions even in translocations having exchanged segments equal in size and the breakpoints located distally to any Giemsa band or other cytological marker. The breakpoints were found to be non-randomly spaced along the chromosomes and their arms. All breaks but one occurred in interband regions of the chromosomes, and none of the breaks was located directly within a centromere. However, short and long chromosome arms recombined at random. An improved tester set of translocations depicting the known break positions of most distal location is presented.     

Physical Mapping of Rice Chromosomes 4 and 7 Using YAC Clones

Physical maps of rice chromosomes 4 and 7 were constructed bylanding yeast artificial chromosomes (YACs) along our high-densitymolecular linkage map. Using 114 DNA markers, 258 individualYACs were located on chromosome 4. Sixty-two out of 258 YACscarried two or more DNA marker positions and formed 16 contigswhich covered a total length of 17.1 cM. The other YACs werearranged to 23 positions. On chromosome 7, 203 individual YACswere landed on 109 DNA markers. Sixty-four out of 203 YACs formed15 contigs which covered a total length of 21.8 cM and 139 YACswere localized to 26 positions. Chromosomes 4 and 7 were coveredwith minimum tiling paths of 45 and 48 YACs, respectively. Takingthe average size of YAC insert DNA to be 350 kb and the entiregenome size to be 430 Mb, about 16–18 Mb of each chromosomeor an estimated 50% of their total lengths have been coveredwith YACs. Physical maps of these 2 chromosomes should be ofgreat help in identifying useful trait genes and unravelinggenetic and biological characteristics in rice.     

Double-strand breaks on artificial chromosomes in yeast

Yeast artificial chromosomes composed primarily of bacteriophage λ DNA exhibit very low levels of meiotic crossing over compared with similarly sized intervals of natural yeast DNA. When these recombinationally quiet chromosomes were augmented with a 12.5 kb insert of sequences from yeast chromosome VIII, genetic studies demonstrated that the artificial chromosomes had acquired recombination properties characteristic of this region of chromosome VIII. On authentic yeast chromosomes, most meiotic recombination events are initiated at sites where the DNA is cleaved to create a double-strand break (DSB). This report describes physical analyses that were carried out to examine the relationship between DSB sites and the recombination behavior of the artificial chromosomes. The results show that DSBs are rare on these artificial chromosomes, except for the 12.5 kb insert. Mapping of the DSB sites shows that their positions correlate with the previously determined positions of DSB sites on chromosome VIII. Deletion of two characterized chromosome VIII DSB sites from the 12.5 kb insert on the artificial chromosome resulted in the loss of the predicted DSB fragments and a reduction in crossing over between artificial chromosomes. Received: 15 May 1998; in revised form: 26 September 1999 / Accepted: 18 November 1999     

A pepsin-revealed material possibly related to chromosomal banding

The enzymes pepsin, alpha-chymotrypsin, trypsin, RNase and DNase were applied to preparations of human metaphase chromosomes before staining to study whether dissociable materials related to the formation of G-, Q- and C-bands would be seen. Treatment with active pepsin but not the other enzymes revealed material with ribonucleo-protein properties which dissociated from the chromosomes and formed a halo.--Lateral extensions from the chromatids stretched to the rim of the halo and appeared at positions corresponding to G-bands. A G-band may be defined as a ring of stable chromatid-matrix binding at positions where the chromatids coil to form lateral extensions.     

Karyotype analysis of Lilium longiflorum and Lilium rubellum by chromosome banding and fluorescence in situ hybridisation.

Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red- and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.     

Genetic Lengths and Break Points in Twelve Chromosomes of GOSSYPIUM HIRSUTUM Involved in Ten Reciprocal Translocations

Chromosome configurations were recorded in about 5500 pollen mother cells (PMC's) in 2n and 2n-1 (missing the intact A-genome chromosome) heterozygotes of ten reciprocal translocations involving six A-genome chromosomes (H1, H2, H3, H4, H6 and H7) and six D-genome chromosomes (H14, H15, H16, H19, H20 and H21) of Gossypium hirsutum. From these records, chiasma frequencies at each of six positions were determined for nine translocations and at two positions for one. These frequencies were used to calculate recombination frequencies in different chromosome regions, and from these distances the breakpoints in 15 chromosome arms were mapped relative to each other and to their respective centromeres, insofar as the data permitted. The karyotype so derived for twelve chromosomes is in reasonably good agreement with data from genetic mapping, telosome and monosome mapping, and the mitotic idiogram.     

Study of the organization of polytene chromosomes in the nuclei of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> pseudonurse cells with the use of the DNA library of the <Emphasis Type="Italic">Drosophila orena</Emphasis> chromocenter

The location of the Drosophila orena chromocenter in polytene chromosomes of pseudonurse cells of the D. melanogaster ovaries (the otu11 mutation) and salivary glands has been studied. Numerous sites of location of the D. orena chromocenter DNA have been found throughout the length of D. melanogaster chromosomes. The specific distribution of the binding sites for the DNA probe has made it possible to identify chromosomes and analyze their mutual positions in the three-dimensional space of the nuclei of pseudonurse cells. The mutual positions of chromosomes have been found to vary, the pericentromeric regions of different chromosomes differing from one another in associative ratios.     

Standard G-, Q-, and R-banded ideograms of the domestic sheep (Ovis aries): homology with cattle (Bos taurus). Report of the committee for the standardization of the sheep karyotype.

Revised G-, Q- and R-banded karyotypes and ideograms for sheep chromosomes at the 420-band level of resolution are presented. The positions of landmark bands on the sheep chromosomes are defined by their distance relative to the centromere to facilitate comparison with equivalent cattle chromosomes. Chromosome-specific (reference) molecular markers that have been mapped to sheep chromosomes and their equivalent cattle chromosomes are proposed. Reference markers will facilitate genome comparisons between sheep and cattle and minimise confusion due to chromosome nomenclature. Numbering of the Robertsonian translocation chromosomes remains as previously reported.     

Chromosome ideograms of the laboratory rat (Rattus norvegicus) based on high-resolution banding, and anchoring of the cytogenetic map to the DNA sequence by FISH in sample chromosomes

A detailed banded ideogram representation of the rat chromosomes was constructed based on actual G-banded prometaphase chromosomes. The approach yielded 535 individual bands, a significant increase compared to previously presented ideograms. The new ideogram was adapted to the existing band nomenclature. The gene locus positions in the rat draft DNA sequence were compared to the chromosomal positions as determined by dual-color FISH, using rat (RNO) chromosomes 6 and 15 and a segment of RNO4 as sample regions. It was found that there was generally an excellent correlation in the chromosome regions tested between the relative gene position in the DNA molecules and the sub-chromosomal localization by FISH and subsequent information transfer on ideograms from measurements of chromosomal images. However, in the metacentric chromosome (RNO15), the correlation was much better in the short arm than in the long arm, suggesting that the centromeric region may distort the linear relationship between the chromosomal image and the corresponding DNA molecule.