Short communication: the cell cycle dependent phosphorylation of histone H3 is correlated with the condensation of plant mitotic chromosomes

Mitotically dividing cells of Secale cereale, Hordeum vulgare and Vicia faba were studied by indirect immunofluorescence using an antibody recognizing phosphorylated histone H3. The study revealed the following features: (i) the H3 phosphorylation starts at prophase and ends at telophase in the pericentromeric chromatin, is associated with the condensation of mitotic chromosomes and is independent of the distribution of late replicating heterochromatin. (ii) Compared with other chromosome regions, the pericentromeric chromatin is histone H3 hyperphos- phorylated. (iii) The study of a semi-dicentric chromo- some revealed that only at intact centromeres is the chromatin hyperphosphorylated at H3.

Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints.

We have developed a new technique for the physical mapping of barley chromosomes using microdissected translocation chromosomes for PCR with sequence-tagged site primers derived from >300 genetically mapped RFLP probes. The positions of 240 translocation breakpoints were integrated as physical landmarks into linkage maps of the seven barley chromosomes. This strategy proved to be highly efficient in relating physical to genetic distances. A very heterogeneous distribution of recombination rates was found along individual chromosomes. Recombination is mainly confined to a few relatively small areas spaced by large segments in which recombination is severely suppressed. The regions of highest recombination frequency (

Integration of microsatellite markers into the translocation-based physical RFLP map of barley chromosome 3H.

PCR with the DNA of translocation chromosomes and marker-specific primers has been used to merge genetically mapped microsatellite (MS) markers into the physically integrated restriction fragment length polymorphism (RFLP) map of barley chromosome 3H. It was shown that the pronounced clustering of MS markers around the centromeric region within the genetic map of this chromosome results from suppressed recombination. This yielded a refinement of the physically integrated RFLP map of chromosome 3H by subdivision of translocation breakpoints (TBs) that were previously not separated by markers. The physical distribution of MS markers within most of the subchromosomal regions corresponded well with that of the RFLP markers, indicating that both types of markers are similarly valuable for a wide range of applications in barley genetics.

Assignment of oat linkage groups to microdissected Avena strigosa chromosomes.

Microdissection of metaphase chromosome preparations of diploid oat Avena strigosa (2n = 14) allowed isolation of the three individual chromosomes with distinct morphologies, numbers 2, 3 and 7. Using a PCR approach based on the DNA of microdissected chromosomes, STS derivatives of RFLP markers, genetically mapped in Avena spp. linkage maps, have been physically assigned to these three chromosomes. Based on either two or four RFLP-derived STS markers, the A. strigosa chromosomes 2 and 3 were found to be homoeologous to the oat linkage groups C and E, respectively. With the DNA of chromosome 7, four RFLP-derived STS markers located within the central part of linkage group F and two distal ends of linkage group G were amplified. Accordingly, chromosome 7 corresponds to linkage group F and, most probably, is involved in an A. strigosa-specific chromosomal translocation relative to the diploid species Avena atlantica and Avena hirtula, of which the cross progeny was used for linkage mapping of the tested RFLP clones.

Differences between genetic and physical centromere distances in the case of two genes for male sterility in barley.

Linkage studies with thirty translocations (one of the two chromosomes involved being number 4) in relation to msg24 (chromosome 4) and thirteen translocations (one of the two chromosomes involved being number 6) in relation to msg6 (chromosome 6) show without exception close linkage for all combinations tested. The results indicate that both genes are located genetically in or close to the centromere regions of their chromosomes.Cytological analysis of two BTT stocks (balanced tertiary trisomics) ascertained the respective chromosome arms (both msg24 and msg6 on the short arms) and revealed marked differences between genetic and physical centromere distances. The reason is obviously the high content of centromeric heterochromatin occupying both the chromosome arms involved.

The physical relationship of barley chromosome 5 (1H) to the linkage groups of rice chromosomes 5 and 10.

Using a recently developed polymerase chain reaction (PCR)-mediated approach for physical mapping of single-copy DNA sequences on microisolated chromosomes of barley, sequence-tagged sites of DNA probes that reveal restriction fragment length polymorphisms (RFLP) localized on the linkage maps of rice chromosomes 5 and 10 were allocated to cytologically defined regions of barley chromosome 5 (1H). The rice map of linkage group 5, of about 135 cM in size, falls into two separate parts, which are related to the distal portions of both the short and long arms of the barley chromosome. The markers on the rice map of chromosome 5 were found to be located within regions of the barley chromosome which show high recombination rates. The map of rice chromosome 10, of about 75 cM in size, on the other hand, is related to an interstitial segment of the long arm of chromosome 5 (1H) which is highly suppressed in recombination activity. For positional cloning of genes of this homoeologous region from the barley genome, the small rice genome will probably prove to be a useful tool. No markers located on rice chromosomes were detected within the pericentric Giemsa-positive heterochromatin of the barley chromosome, indicating that these barley-specific sequences form a block which separates the linkage segments conserved in rice. By our estimate approximately half of the barley-specific sequences of chromosome 5 (1H) show a dispersed distribution, while the other half separates the conserved linkage segments.

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.

Effects of chromosome reconstruction on deletion clustering in Hordeum vulgare.

The barley standard karyotype, two reconstructed karyotypes with all chromosomes interdistinguishable, and four translocation lines were treated with maleic hydrazide. A specific chromosomal site in satellite chromosome 7 (segment 44 adjacent to the nucleolus organizer region) of the standard karyotype was found to represent a deletion hot spot. A sample of specifically reconstructed karyotypes were used to check whether or not transposition of the hot spot region, or changes of its neighborhood, would affect its involvement in deletions. One of the seven karyotypes (translocation line T 505 with a pair of chromosomes having both nucleolus organizer regions and satellites in opposite arms) was without deletion clustering in segment 44. At the same time, a prominent Giemsa band close to the secondary constriction was absent from segment 44. These data show that the involvement in deletions of a certain chromosome segment is modifiable in certain cases by chromosome reconstruction. Similar observations have been made in Vicia faba.

Sequence analysis and gene identification in a set of mapped RFLP markers in barley (Hordeum vulgare).

The "Igri/Franka" (I/F) map ranks among the most comprehensive genetic linkage maps of barley (Hordeum vulgare), containing a large number of markers derived from cDNA and genomic PstI clones. Fourty-three cDNA clones and 259 genomic clones were at least partially sequenced and compared with the major data bases of protein and nucleic acid sequences. Of the cDNA clones, 53% show significant similarity to known sequences in protein data bases. A comparison of sequences from genomic clones to nucleic acid sequence data bases revealed similarities for 9% of the clones. For cDNA sequences analyzed the same way, significant similarities were observed for 35% of the clones. These results show that genomic PstI clones, although containing genes at a significant frequency, represent an inappropriate source for an efficient, systematic gene identification in barley. Sequence information obtained in the context of the present study provides a resource for the conversion of these markers into sequence-tagged site (STS) markers and their use in PCR assays.

Barley chromosome arms longer than half of the spindle axis interfere with nuclear divisions.

We have tested the influence of recombinantly-elongated chromosome arms on nuclear divisions in barley and confirmed a rule according to which half the length of the average spindle axis defines the upper tolerance limit for chromosome arm length. A slightly longer chromosome arm caused incomplete separation of sister chromatids in approximately 70% of mitotic telophase cells and >2.5% of daughter cells showing a micronucleus, due to disruption of non-separated sister chromatids by the newly forming cell wall. In homozygous condition, this elongated chromosome mediated a slower growth and reduced fertility of the carrier plants. Its meiotic transmission was not impaired because of the larger spindle dimensions in meiocytes as compared to those in mitotic cells.

Different chromosomal distribution patterns of radiation-induced interchange breakpoints in barley: first post-treatment mitosis versus viable offspring.

Translocation breakpoints (TBs) induced by ionizing radiation are nonrandomly distributed along barley chromosomes. When first post-treatment mitoses were evaluated, centromeres and the heterochromatin-containing proximal segments tended to be more than randomly involved, and terminal segments to be less than randomly involved in translocations. Contrary to this, small chromosomal regions in median and distal arm positions, characterized by high recombination rates and high gene density, were identified as preferred sites for the origination of viable translocations, probably due to deviations in chromatin organization. Apparently, the position of a TB has an influence on the rate of viability versus elimination of the carrier cells. Surprisingly, TBs within centromeres and heterochromatin-containing segments seem to be more harmful for survival than those induced in gene-rich regions.

'Nucleolar dominance' as observed in barley translocation lines with specifically reconstructed SAT chromosomes.

Diploid homo- and heterokaryotypes of barley translocation lines with only one satellite chromosome pair containing two nucleolus organizer regions (NORs) in opposite arms were found to show repressed nucleolus formation by the transposed NOR as evident from the formation of only micronucleoli. The same was true for auto-tetraploid homokaryotypes and for translocation lines with all NORs tandemly arranged into the same chromosome arm. When NORs were transposed to chromosomes without NOR in the standard karyotype, the normal pattern of nucleolus formation remained unaffected. The modified mode of nucleolus formation after the combination of all NORs in one chromosome pair is interpreted to be due to intrachromosomal nucleolar dominance analogous to interchromosomal nucleolar dominance observed in certain interspecific hybrids.

Polymerase chain reaction mediated localization of RFLP clones to microisolated translocation chromosomes of barley.

A new strategy has been devised and used for the physical localization of genetically mapped restriction fragment length polymorphism (RFLP) clones to barley chromosomes. Morphologically distinct translocation chromosomes from synchronized root-tip meristems were microisolated and their DNA was used as a template for polymerase chain reaction with sequence-specific primers. Four RFLP clones were assigned to cytologically defined segments of chromosome 5. This related approximately one-third of the map length of linkage group 5 to approximately one-fifth of the mitotic metaphase length of chromosome 5. The technique may substantially contribute to the connection of the RFLP-based genetic linkage maps with cytological markers of the barley chromosomes.

Sister chromatid exchanges in barley.

A mean frequency of 20.6 sister chromatid exchanges (SCEs) per cell has been observed in a reconstructed karyotype of Hordeum vulgare by application of the FPG technique after unifilar incorporation of BrdU into chromosomes. The involvement in SCEs of the 48 segments into which the chromosome set had been subdivided was, with a single deviation, length proportional and independent of the segment's heterochromatin content. Asymmetric bands, indicative of an uneven distribution of adenine and thymidine between the DNA strands in adenine (A)-thymidine (T) rich chromosome regions, could not be detected after incubation of the cells in BrdU for one cycle of DNA replication.

Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.).

A high-yield method for isolation of barley chromosomes in suspension, their analysis and sorting using flow cytometry is described. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatment with 2 mmol/L hydroxyurea for 18 h, 2.5 micromol/L amiprophos methyl for 2 h, and ice water (overnight). This treatment resulted in metaphase indices exceeding 50%. Synchronized root tips were fixed in 2% formaldehyde for 20 min and chromosomes were released into a lysis buffer by mechanical homogenization, producing, on average, 5 x 10(5) chromosomes from 50 root tips. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis and sorting. While it was possible to discriminate and sort only one chromosome from a barley cultivar with standard karyotype, up to three chromosomes could be sorted in translocation lines with morphologically distinct chromosomes. The purity of chromosome fractions, estimated after PRINS with primers specific for GAA microsatellites, reached 97%. PCR with chromosome-specific primers confirmed the purity and suitability of flow-sorted chromosomes for physical mapping of DNA sequences.