The hierarchically organized splitting of chromosomal bands for all human chromosomes
© Kosyakova et al; licensee BioMed Central Ltd. 2009
Received: 15 January 2009
Accepted: 26 January 2009
Published: 26 January 2009
Chromosome banding is widely used in cytogenetics. However, the biological nature of hierarchically organized splitting of chromosomal bands of human chromosomes is an enigma and has not been, as yet, studied.
Here we present for the first time the hierarchically organized splitting of chromosomal bands in their sub-bands for all human chromosomes. To do this, array-proved multicolor banding (aMCB) probe-sets for all human chromosomes were applied to normal metaphase spreads of three different G-band levels. We confirmed for all chromosomes to be a general principle that only Giemsa-dark bands split into dark and light sub-bands, as we demonstrated previously by chromosome stretching. Thus, the biological band splitting is in > 50% of the sub-bands different than implemented by the ISCN nomenclature suggesting also a splitting of G-light bands. Locus-specific probes exemplary confirmed the results of MCB.
Overall, the present study enables a better understanding of chromosome architecture. The observed difference of biological and ISCN band-splitting may be an explanation why mapping data from human genome project do not always fit the cytogenetic mapping.
The biological nature of hierarchically organized splitting of bands of human chromosomes remained an enigma since the first banding methods were described in 1970 and 1971. The technique introduced by Lore Zech in Caspersson's laboratory involved quinacrine mustard (Q-banding) and fluorescence microscopy , while other used Giemsa (G-banding) [2, 3]. Though several methods producing chromosome bands were developed later, G-banding became the one most widely used. A uniform system of human chromosomal nomenclature, which allowed to design not only individual chromosomes but also chromosome regions and bands, was proposed for the first time in 1971 at the Fourth International Congress of Human Genetics in Paris , later it developed into the document entitled "An International System for Human Cytogenetic Nomenclature", the last edition of which was published in 2005 . Although recently evolved molecular cytogenetic techniques [6–8] and array-CGH  allow precise characterization of chromosomal abnormalities, analysis of cytogenetic bands is still of great importance. It is often the first step for a clinical diagnosis and in research to understand the biology of an inherited or acquired disease.
The present nomenclature of human chromosomes has been chosen in a more or less randomly manner only by morphological comparison of chromosomal G-bands at different resolution levels and without any systematic investigation about the origin of chromosomal bands. This might be the reason why mapping data from the human genome project do not always fit to the cytogenetic gene mapping data. Thus, an accurate banding nomenclature is required for precise characterization of chromosomal abnormalities.
Recent studies of metaphase chromosomes have revealed that they are remarkably elastic and can be stretched [10, 11]. This extensibility of mitotic chromosomes has been used to increase the resolution of chromosome banding and to do for the first time the systematic analyses of chromosome band splitting [12, 13]. It was found that new sub-bands appeared during the chromosome stretching process and that these sub-bands arose only from G-dark bands. Recently, to confirm these observations we applied another approach which analyzed behavior of multicolor banding (MCB) based pseudo-color bands in respect to G bands on human chromosome 5 using chromosome preparations of different length . Here, we extended and complemented these studies and present for the first time the biologically based hierarchically organized splitting of chromosomal bands for all human chromosomes.
Results and discussion
Differences of ideograms used here compared to those in ISCN 2005:
No splitting of 11p16.1 and 11q14.1 at this stage
As noted by Kowalska and coworkers (2007)  sub-band information provided in different human genome sequence databases are not identical. This might be partially due to the different initial sources of G-banded ideogram used and leads to discrepancies in gene mapping in different databases. ISCN ideograms are used in the NCBI Human Genome browser http://www.ncbi.nlm.nih.gov/ and are based on morphological comparison of chromosomes at different G-band resolution level. At the same time UCSC http://genome.ucsc.edu/ and Ensembl genome browsers http://www.ensembl.org/index.html are based on the ideograms resulted from so called cytogenetic band prediction . The latter method employed results from 9500 FISH experiments to approximate the locations of the 850 high-resolution bands, and thus, could define chromosome band lengths more precisely. Surprisingly, by applying this cytogenetic band prediction algorithm, it was shown that the lengths of the darkest G bands were consistently underestimated, while the opposite was true for the light bands. This finding might be also an indirect proof for the observation that only G-dark bands split into new G-dark and G-light sub-bands, the first ones containing hence potentially higher condensed DNA.
Finally, the present study was only in part able to enlighten the chromosomal architecture of the pericentromeric heterochromatin. It could be shown, that centromere-near subbbands arise e.g. from 1q12, 19q12, 20p11 and 20q11 but not from 9q12 or 16q11.2. Also no new information could be obtained for the heterochromatic short arms of the acrocentric chromosomes. So, neither current genome databases nor cytogenetic nomenclature attempts do consider the DNA or chromatin base of these still somehow enigmatic chromosomal structures.
The biological way of band splitting in peripheral blood lymphocytes is shown for each chromosome in Fig. 4 and for each chromosome ' [see Additional files 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24]. Note: these ideograms are NOT intended to replace neither ISCN ideograms nor nomenclature!
In summary, we achieved now a better understanding of the chromosomal architecture (Fig. 4). The biologically correct nomenclature of sub-bands was found to be in more than 50% different from the ISCN 2005 nomenclature. This may help explaining why mapping data from the human genome project do not always fit the cytogenetic gene mapping data.
Metaphase preparations were done from normal human peripheral lymphocyte cultures. To study chromosomes of different length, different cultivating protocols were applied to obtain metaphases with 300–400, 550 and 850 bands per haploid karyotype. Chromosomes at 300–400 band stages were prepared by harvesting cultures using standard cytogenetic methods . Methotrexate mediated cell culture synchronization  was done to prepare chromosomes at 550-band stage. In order to obtain chromosome preparations at 850-band level we used Synchroset (Euroclone), in combination with Chromosome Kit P (Euroclone) and Buffered Hypotonic Solution (ProCell Reagents).
Multicolor banding (aMCB)
Recently BAC-array mapped aMCB probe sets  for all human chromosomes were applied according to standard protocols [24, 25]. For evaluation of the fluorescence in situ hybridization (FISH) results, ISIS software (MetaSystems, Altlussheim, Germany) was used acc. .
Analysis of aMCB
Using ISIS software (MetaSystems, Altlussheim, Germany) chromosome region-specific fluorescence profiles can be converted into computer-based pseudo-colors. One pseudo-color band corresponds to a specific fluorochrome combination and (in parts) to a specific fluorochrome intensity, which can be variably assigned to any resolution level. As stated in , pseudo-color schemes with different number of pseudo-colors were created at the G-stage of 850 bands, and then applied to chromosomes of different length. When limited number of pseudo-colors is used, aMCB pattern remains stable irrespective of the chromosome condensation . But if higher numbers of pseudo-colors are assigned, then disappearance of some pseudo-colors was observed on middle (550 bands stage) and short (300–400 bands stage) chromosomes. 10 copies of each chromosome at each band-stage level were evaluated; the evaluation process followed the rules suggested previously by .
BAC clones, RP11-876B11, RP11-673C15, RP11-904G16, RP11-458F8, RP11-584N20, RP11-243C20 were purchased from Sanger Centre, UK http://www.sanger.ac.uk/ DNA preparations from cultured bacteria (containing required construct) were performed using the QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer's protocol. DNA was amplified using DOP-PCR with three low-temperature cycles, and then labelled with biotin-16-dUTP, SpectrumGreen-dUTP or SpectrumOrange-dUTP by label-PCR [25, 26]. Biotin-16-dUTP labeled probes were detected with Streptavidin-Cy5. Unincorporated nucleotides were removed by ethanol precipitation. BACs for the same chromosome were hybridized in parallel to chromosome preparations of different length.
Supported in parts by the DFG (LI820/11-1, 436 RUS 17/135/03, 436 RUS 17/109/04, 436 RUS 17/22/06, WE 3617/2-1) and the Evangelische Studienwerk e.V. Villigst.
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