Chromosome Banding Techniques Research Articles

Robertsonian polymorphism in the marine gastropod, Nucella lapillus: advances in karyology using rDNA loci and NORs

Previous studies of the Robertsonian polymorphism in the Atlantic dog-whelk, Nucella lapillus (2n = 26-36), have been limited by the inability to identify unequivocally individual chromosomes in the karyotype. This species, as with many other marine invertebrates, has proven largely refractory to the standard (mammalian) chromosome-banding techniques. In this study, fluorescence in situ hybridization (FISH) using a rDNA probe was applied to the metaphase chromosomes of the 2n = 26 and 2n = 36 forms of N. lapillus. The results were compared with silver-staining of the nucleolar organizer regions (NORs). The FISH technique was shown to be more sensitive and less intrinsically prone to variation than the silver-staining method. An additional NOR/rDNA locus was observed in the 2n = 36 form which, to date, has not been seen in any 2n = 26 population. The 2n = 36 karyotype is described for a south-west UK population that differs from that reported previously in the literature. After fission, Robertsonian metacentrics are shown to correspond to at least one subtelocentric product.

Cytogenetics and Molecular Genetics of Bone and Soft Tissue Tumors

Publisher Summary This chapter focuses on the cytogenetic and molecular genetics of bone and soft tissue tumors. The majority of cytogenetic data stem from the hematological malignancies, and only slightly more than 25% of the more than 22,000 cases reported with chromosome aberrations identified by chromosome banding techniques represent solid tumors. Among these, the bone and soft tissue tumors (BSTT) belong to one of the cytogenetically best characterized groups of tumors. The first reports on consistent tumor-associated chromosome aberrations in BSTT are on Ewing sarcomas. Characteristic chromosome changes have been described in benign and malignant BSTT as well as in some lesions whose true neoplastic nature is uncertain and debated. The finding of specific chromosome aberrations in a number of BSTT has provided molecular geneticists with important information in their efforts to investigate the underlying gene rearrangements. The introduction of new techniques (“molecular cytogenetics”), such as various types of fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), bridges the gap between cytogenetics and molecular genetics and has also opened up new possibilities to investigate genomic rearrangements and has already yielded interesting results. The genetic analysis of BSTT has become increasingly important in diagnostic work, whereas its role in prognostication is still limited. In the chapter, some mesenchymal tumors that are not literally BSTT, such as uterine leiomyomas and chondroid hamartomas of the lung, are dealt with, but other types—such as meningiomas and mesotheliomas—are not addressed. The chapter also discusses aberrations shared by several tumor types. In the diagnostic situation, cytogenetic and/or molecular genetic analysis adds important, sometimes decisive, information to the cytological, histopathological, and electron microscopic examinations of BSTT.

Numerical aberrations of chromosomes 1 and 7 in renal cell carcinomas as detected by interphase cytogenetics.

Alcohol-fixed single cell suspensions of 37 renal cell carcinomas (RCCs) were assessed by both flow cytometry (FCM) and the fluorescence in situ hybridization (FISH) technique, using chromosome 1- and chromosome 7-specific centromere DNA probes. DNA diploidy or near-diploidy was observed in 30 of the 37 RCCs and only 12 of these (near-)diploid tumours were disomic for both chromosomes 1 and 7. Numerical aberrations of chromosome 1 and/or chromosome 7 were present in 18 of the 30 (near-)diploid RCCs and five of these cases showed monosomy for chromosome 1 in more than 50 per cent of the tumour cells. A double target FISH, with a centromeric and a telomeric specific probe for 1p36, excluded misinterpretation on the basis of clustering of 1q12, and suggested a complete loss of chromosome 1. All these five (near-)diploid RCCs with monosomy for chromosome 1 were eosinophilic chromophilic cell carcinomas, according to the Thoenes classification of RCC. This observation is of special interest, because it was recently concluded from cytogenetic studies that the diagnosis of chromophilic renal cell carcinoma must be considered as obsolete. Monosomy for chromosome 1 seems to be a non-random numerical aberration of (near-)diploid eosinophilic chromophilic cell carcinomas, and a gain of one or more chromosomes 1 appeared to be a common phenomenon in RCCs, especially in the DNA aneuploid tumours. As these chromosomal abnormalities were not found in the earlier classical cytogenetic studies, we conclude that in situ hybridization techniques are required in addition to chromosome banding techniques to obtain a complete characterization of the chromosome imbalances in RCCs.

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.

Chromosome Banding Techniques

Chromosome banding techniques produce a series of consistent landmarks along the length of metaphase chromosomes that allow for both recognition of individual chromosomes within a genome and identification of specific segments of individual chromosomes. These landmarks facilitate assessment of chromosome normalcy, identification of sites of chromosome breaks and alterations, and location of specific genes. This unit covers these basic banding techniques (Q-banding, G-banding, and R-banding), which produce virtually identical patterns of bands along the length of human chromosomes, although the bands and polymorphic regions highlighted may differ with each technique. These techniques highlight reproducible landmarks along the length of the chromosome and specialized staining techniques can be used to highlight particular regions of chromosomes, such as heterochromatic and repeated-sequence segments. These specialized techniques, nucleolar organizer region (NOR) staining, centromeric heterochromatin staining (C-banding), methylated satellite DNA staining (distamycin-DAPI banding), and replication banding are also presented in this unit.

Chromosome banding and identification. Fluorescence.

General characteristics of chromosome banding and banding techniques have been described in the previous chapter ( Chapter 6). In general, fluorescence banding provides much the same information as banding using absorption staining, but fluorescence staining methods are generally simpler than banding methods using absorption staining and, in many respects, are more reliable. On the other hand, fluorescent preparations are not permanent and have to be examined using a fluorescence microscope. As well as providing the same general information as banding with absorption staining, most fluorescence banding methods also provide specific types of information (for example, on heteromorphisms of heterochromatin) that are not shown by other methods. Details of these applications will be given under the specific methods.

Mitotic metaphase chromosome preparation from peripheral blood for high resolution.

AbstractThe advent of chromosome banding techniques some 20 years ago (1,2) allowed the unequivocal identification of every chromosome in the human metaphase and provided a mapping scheme along each chromosome. Subsequently, a great deal of research has centered on preparing longer chromosomes with more bands visible. Chromosomes condense as they move through mitosis, and adjacent bands close up and appear to fuse. The earlier stages are longer with more bands recognized. It is not always possible to define the mitotic stage of a particular cell. International standards have been agreed for various numbers of bands in the haploid set. Thus we have 400-, 550-, and 850-band sets (3). Other workers report the use of even longer chromosomes (4,5). High-resolution banding has undoubted advantages in many fields. As well as allowing greater accuracy in traditional karyotype analysis, there are many reports of microdeletions and other abnormalities detected only on extended chromosomes (6). Likewise, in situ hybridization and gene localization techniques are taking advantage of the improved resolution.KeywordsHypotonic SolutionRelease AgentResuspend CellLonge ChromosomeUndoubted AdvantageThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

High resolution mapping of the MYCN proto-oncogene at human chromosome 2p24.3 by fluorescence in situ hybridisation

The MYCN proto-oncogene was previously mapped to human chromosome 2p24.1 by analysis of mouse x human somatic cell hybrids and radioactive in situ hybridisation to normal human chromosomes. However, using fluorescence in situ hybridisation (FISH) and high resolution chromosome banding techniques MYCN has been reassigned to 2p24.3.

Analysis of chromosomes with restriction endonucleases and DNase hypersensitivity.

The introduction of chromosome banding techniques about 20 years ago has not only provided a very powerful tool for chromosome identification, but has also highlighted structural and functional differences between different chromosomal segments (). Restriction endonucleases (RE) have been widely used to study some aspects of chromosome organization (, , ). The original methods, involving digestion followed by Giemsa or ethidium bromide (EB) staining, lack both specificity and sensitivity. It is now clear that the Giemsa/EB staining does not necessarily represent the distribution of restriction sites along the chromosomes; other factors, such as the accessibility of the REs to the targets or the extractability of the DNA fragments produced, need to be considered.

Rapid detection of 1B, 1BL/1RS heterozygotes in the development of homozygous 1BL/1RS translocation stocks of Triticum turgidum (2n = 4x = 28)

A biochemical marker was utilized to facilitate detection of chromosome 1B, 1BL/1RS translocation heterozygote plants in segregating backcross progenies during the development of 1BL/1RS homozygous lines in several Triticum turgidum L. cultivars (2n = 4x = 28; AABB). Isoelectric focussing of glucose phosphate isomerase (GPI) on either pH 3.5-9.5 or 5.5-8.5 polyacrylamide gels facilitated the detection of 1B, 1BL/1RS translocation heterozygotes from the homozygous 1B or 1BL/1RS derivatives during each backcross of the heterozygote to the respective recurrent parent. The biochemical diagnostic procedure complements the more time consuming and cumbersome chromosome banding technique. This GPI diagnostic in durum 1BL/1RS development is also swifter than a similar stocks development in T. aestivum where both GPI and acid PAGE are essential.

A supernumerary chromosome segment in Locusta migratoria

A single female of Locusta migratoria was found to be heterozygous for a supernumerary heterochromatic segment distally located on the M6 autosome close to its nucleolus organiser region (NOR). Reactions to several chromosome banding techniques revealed its heterochromatic nature and its composition of GC-rich DNA sequences and likewise the NORs in this species. This suggests an origin for the extra segment by amplification of GC-rich DNA sequences contained in the distal NOR of the M6 chromosome, which is reinforced by the observation that the NOR of segmented M6 chromosomes produced the larger nucleolus in embryo prophase cells, such as would be expected from the presence of rRNA genes in the extra segment. No accumulation mechanism was detected in this female after analyzing the 213 embryo offspring produced, but an increase in the number of nucleoli per interphase nucleus was noted in heterozygous embryos in respect to standard homozygous ones.

Fluorescent banding pattern analysis of eight taxa of Phaseolus and Vigna in relation to their phylogenetic relationships

Phylogenetic relationships among eight taxa of seven species of Phaseolus and Vigna (Phaseolus angularis, P. aureus, P. calcaratus, P. coccineus, P. vulgaris, Vigna sesquipedalis and V. sinensis; 2n = 22 each) were studied by the fluorescent chromosome banding technique. Preparations of somatic metaphase chromosomes of each taxon were sequentially stained with Giemsa, GC-specific fluorochrome chromomycin A3 (CMA) and AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). On the basis of the fluorescent banding patterns of the 22 chromosomes of each taxon, P. angularis, P. coccineus (from China and Korea) and P. vulgaris were grouped into one group ("Phaseolus group"), P. aureus and two Vigna species were grouped into another ("Vigna group") and P. calcaratus was grouped in an independent group.

Growth and developmental patterns in Prader-Willi syndrome.

Eleven Japanese patients with Prader-Willi syndrome were studied from infancy, and examined with respect to their growth and development. The chromosomal aberration was observed in 40% of the patients using a high-resolution chromosome banding technique. The birth weight in 10 out of the 11 patients was below the mean and the rate of body weight gain was severely retarded by 6 months in all cases, which suggested an insufficient utilization of nutrients in uterine life and in early infancy. The rate of height increase as well as that of body weight increase were also transiently retarded from 10 to 18 months of age in eight cases, and the body weight gain increased dramatically after 10-18 months due to hyperphagia. The patients' mean milestones of development were delayed (the mean developmental quotient was 50) in comparison with those of control patients; there was no difference between the developmental milestones of the two groups with and without the chromosomal aberration.

Chromosome banding patterns in Lettuce species (Lactuca sect.Lactuca, Compositae)

Chromosome banding patterns obtained with C- and N- banding, and AgNO3 staining were studied in somatic metaphase complements of fourLactuca species.L. sativa andL. serriola have almost identical chromosome morphology, andL. saligna differs only slightly from them, butL. virosa is quite distinct from the other species. A gross comparison of the banded karyotypes suggests a closer relationship ofL. saligna toL. sativa/serriola than toL. virosa. Our data agree with the results of previous crossing experiments in these species but conflict partly with recent RFLP data which indicate a closer phenetic relationship ofL. saligna toL. virosa than toL. sativa/serriola. Such a discrepancy may be explained assuming that domestication ofL. sativa/serriola resulted in an increased selection pressure on unique DNA sequences as demonstrated by the RFLP data. Differential evolution of specific heterochromatin classes (and presumably of highly repetitive DNA classes), as revealed by chromosome banding techniques was not linked to domestication. Thus the disparity in conclusions about relationship (in terms of genetic similarity) as based on the different experimental approaches reflects a non-parallel evolution of highly repetitive vs. unique DNA classes.

Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that “niches” need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

Long-Term Engraftment of Normal and Post-5-Fluorouracil Murine Marrow Into Normal Nonmyeloablated Mice

AbstractWe report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that “niches” need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 × 106 male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU–pretreated male marrow as shown 1 to 12 months posttransplant in noncytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1 % to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post–5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post–5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

Restriction endonucleases: Powerful tools to induce chromosome markers

The introduction of chromosome banding techniques about twenty years ago provided a simple and reliable method for chromosome identification, as well as highlighting structural and functional differences between chromosomal segments. On this basis, it has been possible to identify different chromosome markers and to analyse the extent of variation among different taxa at the chromosome level. Since 1968 when the first banding technique was employed, other more sophisticated methods have now become available which permit a deeper understanding of banding mechanisms down to the molecular level. Recently, techniques involving restriction endonucleases have been developed (REs) which may recognise specific nucleotide sequences along the chromosomes. Because of the high specificity of these enzymes, their in situ application on fixed chromosomes followed by Giemsa or Ethidium Bromide staining opens the possibility of obtaining a very large number of different banding patterns. To date, the main limitation of this technique lies in certain structural aspects involving the accessibility of REs to DNA, or chromatin extraction. More recently, yet another method with increased sensitivity and specificity has become available. In this method, sites of RE attack on chromosomes are identified by ‘nick translation’ (RE/NT) inducing banding patterns that do not depend on DNA extraction. Both applications assayed on several organisms have provided new chromosome markers as well as valuable information on structural and functional aspects.

High catabolism of BrdU may explain unusual sister chromatid differentiation and replication banding patterns in cancer cells

Two T-cell acute lymphoblastic leukemia (ALL) cell lines, PEER and CCRF-CEM, were studied by various chromosome banding techniques, including 5-bromodeoxyuridine (BrdU) incorporation methods. Although of very similar origin, these two cell lines behave quite differently. In particular, CEM cell line exhibited an abnormal replication banding pattern (RBP) and poor sister chromatid differentiation (SCD). Study of their thymidylate synthase and thymidine kinase activities indicated that CEM had a more active salvage pathway for thymidylate synthesis than did PEER cell line, which may suggest an efficient BrdU incorporation and its fast decrease in culture medium, resulting in the observed peculiarities. However, this was contradictory to the fact that CEM need a higher dose of BrdU than do PEER cells to induce SCD and RBP. Finally, the radioactivity from 3H-thymidine decreased in the culture medium much faster for PEER cell line than for CEM cell line, and about 50% of the remaining radioactivity was due to 3H-thymidine for CEM cell line. Thus, the abnormal SCD and RBP are explained by an active catabolism of thymidine and BrdU in CEM cell line.

Karyotype differentiation and chromosomal variability in springtails (Collembola, Insecta)

Karyological studies in the mitotic complements of arthropleonic springtails (Collembola) were carried out, applying chromosome-banding techniques (Giemsa C banding) for the first time. These techniques provide important indications on karyological variability and the genetic heterogeneity of local populations. Karyotype architecture may be of taxonomic significance at the species level. Chromosomal studies in various populations of the group Onychiurus armatus (Tullb.) high-light the importance of future karyological studies, especially in taxonomically complicated groups.

Multiple fluorescence in situ hybridization

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.