The use of subchromosome-length unique band sequences in the analysis of prophase chromosomes.

Using human prophase chromosome ideograms at the 850-band stage, we previously demonstrated that the 24 prophase ideograms can be divided into a set of 94 unique band sequences, each having a recognizable banding pattern distinct from other nonhomologous chromosome portions. Using actual prophase mitotic cells in this study, we analyzed the p arm of chromosome 11 and of chromosomes 16-22 and characterized a similar set of unique band sequences on actual chromosomes. This set of unique band sequences, a statistical comparison scheme, and image-processing techniques outlined in the present report can be used to identify and distinguish banding patterns of these chromosomes and to determine band pattern abnormalities.     

Analysis of high-resolution R-bands, obtained by heat-denaturation and Giemsa staining, on human prophase chromosomes

RHG-bands (heat-denatured Giemsa R-bands) of human prophase chromosomes were analyzed at high resolution, and the banding patterns at prophase and metaphase are presented. The bands were compared with those of the International Standard Cytogenetic Nomenclature idiograms and of the G-band idiograms proposed by J. J. Yunis. The number, size, and position of the RHG-bands correspond rather well with their equivalent G-negative bands, but some differences were noted in the zones of preferential stretching, the juxtacentromeric regions, and the telomeres. Variations in the centromere index and the banding pattern in heterochromatin were also discussed.     

Towards quantitative cytotaxonomy of primates: Idiogram construction of great ape chromosomes by «fourier-warping»

To faciliate quatitative comparative cytogenetics of primate chromosomes we present a new type of idiogram from human and great ape chromosomes. These idiograms were obtained by computer densitometry and «Fourier-Warping» (FW) as described elsewhere (Maurier & Wienberg, 1991). In contrast to published idiograms, the «FW-idiograms» represent the mean position, width and density of each band and the mean resolution of the chromosome sample under investigation. In species that deverged about 6–12 my we were able to extract the representative banding features and to demonstrate conservation and change in the karyotype of human and great apes.     

Reproducible but dynamic positioning of DNA in chromosomes during mitosis

How DNA is folded into chromosomes is unknown. Mitotic chromosome banding shows reproducibility in longitudinal compaction at a resolution of several megabase pairs, but it is less clear whether DNA sequences are targeted laterally to specific locations. The in vitro chromosome assembly of prokaryotic DNA suggests that there is a lack of sequence requirements for chromosome condensation, implying an absence of DNA targeting. Protein extraction experiments indicate, however, that specific DNA sequences may bind to a chromosome scaffold. Chromosome banding patterns, using dyes with differential sequence specificity, have been interpreted to result from the alignment of AT-rich sequences in a partially helically folded chromosome scaffold. But fluorescence in situ hybridization experiments, perhaps owing to technical limitations, have shown at best only slight deviation from a random, lateral sequence distribution. Here we show that there is highly reproducible targeting of specific chromosome segments to the metaphase chromatid axis, but that these segments localize to the periphery of prophase and telophase chromosomes. Unfolding intermediates during anaphase and telophase suggest that sequence repositioning occurs through the global uncoiling of an underlying chromatid structure.     

水稻染色体G—带的研究

用改良的ASG法首次在籼稻(O.sativa subsp.indica)品种珍汕97和粳稻(O.subsp.iaponica)品种秀岭的有丝分裂染色体上显示了G-带,并作了相应的G-带核型分析。就同一材料来说,随着有丝分裂时期的推进,染色体上带纹数目逐渐减少。籼、粳亚种间相对应的同源染色体上G-带带纹特征彼此相似。讨论了水稻G-带带型与染色体不同区域分化的关系;G-带带型与籼、粳稻分歧的关系;以及G-显带的方法。     

Early and late replicative chromosomal banding patterns of Gallus domesticus.

Early and late replicating chromosomal banding patterns of Gallus domesticus were investigated by cell synchronization and incorporation of 5'-bromodeoxyuridine during early and late DNA synthesis. The early replicating chromosomal banding patterns observed, as revealed by either acridine orange or Hoechst 33258/propidium iodide staining, were similar to the structural G-banding patterns obtained by trypsin digestion and Giemsa staining. Late replicating chromosomal banding showed extensive reverse band complementarity to the G-banding pattern. Cell synchronization increased the number of prometaphase and metaphase plates available for analysis. G-banding obtained by Hoechst 33258/propidium iodide staining was investigated due to the fact that it is compatible with chromosomal in situ hybridization procedures that use nonisotopically-labeled DNA probes. Standard replicative G-banded and R-banded idiograms, as obtained after cell synchronization, are proposed.     

多色-FISH技术的进展及其在分子生物医学上的应用

传统显带分析技术以每条染色体独特的显带带型为依据,提供染色体形态结构的基本信息,用于染色体核型的初步分析。然而有些染色体重排由于涉及的片断太小或具有相似的带型,用该方法难以探测或准确描绘。多元荧光原位杂交(M-FISH),光谱核型分析(SKY),FISH-显带分析技术是染色体特异的多色荧光原位杂交技术(mFISH)。它们能够探测出传统显带分析不能发现的染色体异常,提供更准确的核型。M-FISH和SKY均以组合标记的染色体涂染探针共杂交为基础,二者的不同在于观察仪器和分析方法上。它们可对中期染色体涂片进行快速准确分析,描绘复杂核型,确认标记染色体,主要用于恶性疾病的细胞遗传学诊断分析。FISH-显带分析技术以FISH技术为基础,能同时检测多条比染色体臂短的染色体亚区域。符合该定义的FISH-显带分析技术各有特点,其共同特点是都能产生DNA特异的染色体条带。这些条带有更多色彩,能提供更多信息。FISH-显带分析技术已经成功地被用于进化生物学、放射生物学以及核结构的研究,同时也被用于产前、产后以及肿瘤细胞遗传学诊断,是很有潜力的工具。     

Molecular definition of high-resolution multicolor banding probes: first within the human DNA sequence anchored FISH banding probe set.

Fluorescence in situ hybridization (FISH) banding approaches are standard for the exact characterization of simple, complex, and even cryptic chromosomal aberrations within the human genome. The most frequently applied FISH banding technique is the multicolor banding approach, also abbreviated as m-band, MCB, or in its whole genomic variant multitude MCB (mMCB). MCB allows the differentiation of chromosome region-specific areas at the GTG band and sub-band level and is based on region-specific microdissection libraries, producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) mapped, comprehensive, genome-wide human MCB probe set. All 169 region-specific microdissection libraries were characterized in detail for their size and the regions of overlap. In summary, the unique possibilities of the MCB technique to characterize chromosomal breakpoints in one FISH experiment are now complemented by the feature of being anchored within the human DNA sequence at the BAC level.     

Electron microscopy of gold-labeled human and equine chromosomes

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.     

Mapping G-bands on human prophase chromosomes.

OHNUKI's method for demonstrating coils in human metaphase chromosomes also reveals a fine G-band pattern on prophase chromosomes of sufficient clarity to justify an attempt at mapping. Maps are provided for each chromosome to show the maximum number of prophase bands observed, and an intermediate stage in chromosome contraction, tracing the pathways of apparent band fusion as the cell progresses to metaphase, is presented. The prophase bands on many chromosomes tend to occur in distinct groups, the members of which ultimately merge to give the dark G-bands of metaphase chromosomes. Every G-band of the standard metaphase chromosomes. Every G-band of the standard metaphase pattern is compounded from two or more prophase bands. In at least contracted prophase chromosomes examined, some bands are seen which have no obvious metaphase counterpart. There are marked similarities between banded prophases and the chromoomere pattern seen at meiotic prophase. However, since chromosome contraction is a dynamic process, agreement between maps will be expected only for corresponding degrees of chromosome contraction.     

Evolutionary divergence of human chromosome 9 as revealed by the position of the ABL protooncogene in higher primates

Attempts to solve the fundamental questions regarding the descent of man are dogged by superstitions and unexamined orthodoxies. The origin of humans, established a decade ago based upon cytological analysis of ape chromosomes, continues to be called into question. Although molecular methods have provided a framework for tracing the paths of human evolution, conclusive evidence remains elusive. We have used a single ABL gene probe derived from human chromosome 9 to assess the direction of change in the equivalent ape chromosomes. This approach has resulted in a few surprises which again challenge the prevailing view of early primate evolution based solely on chromosome banding patterns. The ABL protooncogene is present on human chromosome 9 at band q34. Similar DNA sequences presumed to represent an ABL gene, are present on chromosome 11 in chimpanzee (Pan troglodytes) but at a different relative location, indicating that the mechanism of the origin of human chromosome 9 is far more complex than has previously been suggested. Nevertheless, in gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus), the equivalent to human chromosome band 9 q34 is apparently located on chromosome 13 at a putative telomeric position and no discernible differences could be established. Despite the presence of the ABL protooncogene on human equivalent ape chromosomes, molecular systematics will continue to generate enigmas in the evolutionary context until the entire genome is sequenced.     

Selenium is involved in the negative regulation of the expression of selenium-free [NiFe] hydrogenases in Methanococcus voltae

Attempts to solve the fundamental questions regarding the descent of man are dogged by superstitions and unexamined orthodoxies. The origin of humans, established a decade ago based upon cytological analysis of ape chromosomes, continues to be called into question. Although molecular methods have provided a framework for tracing the paths of human evolution, conclusive evidence remains elusive. We have used a single ABL gene probe derived from human chromosome 9 to assess the direction of change in the equivalent ape chromosomes. This approach has resulted in a few surprises which again challenge the prevailing view of early primate evolution based solely on chromosome banding patterns. The ABL protooncogene is present on human chromosome 9 at band q34. Similar DNA sequences presumed to represent an ABL gene, are present on chromosome 11 in chimpanzee (Pan troglodytes) but at a different relative location, indicating that the mechanism of the origin of human chromosome 9 is far more complex than has previously been suggested. Nevertheless, in gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus), the equivalent to human chromosome band 9 q34 is apparently located on chromosome 13 at a putative telomeric position and no discernible differences could be established. Despite the presence of the ABL protooncogene on human equivalent ape chromosomes, molecular systematics will continue to generate enigmas in the evolutionary context until the entire genome is sequenced.     

Identification and designation of telocentric chromosomes in barley by means of Giemsa N-banding technique

Summary Seven complete chromosomes and nine telocentric chromosomes in telotrisomics of barley (Hordeum vulgare L.) were identified and designated by an improved Giemsa N-banding technique. Karyotype analysis and Giemsa N-banding patterns of complete and telocentric chromosomes at somatic late prophase, prometaphase and metaphase have shown the following results: Chromosome 1 is a median chromosome with a long arm (Telo 1L) carrying a centromeric band, while short arm (Telo 1S) has a centromeric band and two intercalary bands. Chromosome 2 is the longest in the barley chromosome complement. Both arms show a centromeric band, an intercalary band and two faint dots on each chromatid at middle to distal regions. The banding pattern of Telo 2L (a centromeric and an intercalary band) and Telo 2S (a centromeric, two intercalary and a terminal band) corresponded to the banding pattern of the long and short arm of chromosome 2. Chromosome 3 is a submedian chromosome and its long arm is the second longest in the barley chromosome complement. Telo 3L has a centromeric (fainter than Telo 3S) and an intercalary band. It also shows a faint dot on each chromatid at distal region. Telo 3S shows a dark centromeric band only. Chromosome 4 is the most heavily banded one in barley chromosome complement. Both arms showed a dark centromeric band. Three dark intercalary bands and faint telomeric dot were observed in the long arm (4L), while two dark intercalary bands in the short arm (4S) were arranged very close to each other and appeared as a single large band in metaphase chromosomes. A faint dot was observed in each chromatid at the distal region in the 4S. Chromosome 5 is the smallest chromosome, which carries a centromeric band and an intercalary band on the long arm. Telo 5L, with a faint centromeric band and an intercalary band, is similar to the long arm. Chromosomes 6 and 7 are satellited chromosomes showing mainly centromeric bands. Telo 6S is identical to the short arm of chromosome 6 with a centromeric band. Telo 3L and Telo 4L were previously designated as Telo 3S and Telo 4S based on the genetic/linkage analysis. However, from the Giemsa banding pattern it is evident that these telocentric chromosomes are not correctly identified and the linkage map for chromosome 3 and 4 should be reversed. One out of ten triple 2S plants studied showed about 50% deficiency in the distal portion of the short arm. Telo 4L also showed a deletion of the distal euchromatic region of the long arm. This deletion (32%) may complicate genetic analysis, as genes located on the deficient segment would show a disomic ratio. It has been clearly demonstrated that the telocentric chromosomes of barley carry half of the centromere. Banding pattern polymorphism was attributed, at least partly, to the mitotic stages and differences in techniques.Contribution from the Department of Agronomy and published with the approval of the Director of the Colorado State University Experiment Station as Scientific Series Paper No. 2730. This research was supported in part by the USDA/SEA Competitive Research Grant 5901-0410-9-0334-0, USDA/ SEA-CSU Cooperative Research Grant 12-14-5001-265 and Colorado State University Hatch Project. This paper was presented partly at the Fourth International Barley Genetics Symposium, Edinburgh, Scotland, July 22–29, 1981     

High resolution multicolor-banding: a new technique for refined FISH analysis of human chromosomes.

A new multicolor-banding technique has been developed which allows the differentiation of chromosome region specific areas at the band level. This technique is based on the use of differently labeled overlapping microdissection libraries. The changing fluorescence intensity ratios along the chromosomes are used to assign different pseudo-colors to specific chromosome regions. The multicolor banding of human chromosome 5 is presented as an example.     

High-resolution analysis of DNA replication domain organization across an R/G-band boundary.

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.     

Toward a multicolor chromosome bar code for the entire human karyotype by fluorescence in situ hybridization

A colored banding pattern for human chromosomes is described that distinguishes each chromosome in a single fluorescence in situ hybridization with a set of subregional DNA probes. Alu/polymerase chain reaction products of various human/rodent somatic cell hybrids (fragment hybrids) were pooled into two probe sets that were labeled differentially and detected by red and green fluorescence. Chromosome regions hybridized by DNA present in both pools appeared yellow. The result was a multi-color set of 110 distinct signals per haploid chromosome set for the human karyotype. Each individual chromosome showed a unique sequence of signals, a result termed the “chromosome bar code”. The reproducibility of the hybridization pattern in various labeling and hybridization experiments was analyzed by computer densitometry. We have applied the chromosome bar code both in diagnostic cytogenetics and in genome studies. The approach allows the rapid identification of chromosomes and chromosome rearrangements. Although not yet showing the resolution of classical banding patterns, the present experiments demonstrate various applications in which the present multi-color bar code can significantly add to the spectrum of cytogenetic techniques. Received: 18 December 1996 / Accepted: 10 February 1997     

Chromosomal Assignment of Human Nuclear Envelope Protein Genes LMNA, LMNB1, and LBR by Fluorescencein SituHybridization

We have used fluorescencein situhybridization to establish precise chromosomal localizations for three human genes encoding four different nuclear envelope proteins. Lamin A/C (LMN1, HGMW-approved symbol LMNA) mapped to 1q21.2–q21.3, with a most probable gene assignment to 1q21.3; lamin B receptor (LBR) was localized to 1q42.1; and lamin B1 (LMNB1) was mapped to the interface of bands 5q23.3–q31.1. Assignments were determined by direct placement of signals relative to high-resolution DAPI or G-bands. Comparison of these results of band positions predicted from fractional length measurements to signal placement indicated that more accurate predictions are made using Francke idiograms and that measurement strategy avoids variance due to polymorphic chromosome segments.     

Spectral karyotyping analysis of human and mouse chromosomes

Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.