Centric Fusion

J. Rubes , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Abstract

Different organisms have different sets of chromosomes. Changes in chromosome number, size, and morphology result from various rearrangements such as translocations, inversions, duplications, and centric or tandem fusions. Centric fusion is a very common evolutionary change in which there is a fusion of two nonhomologous chromosomes with terminal or near-terminal centromeres that produce bi-armed submetacentric or metacentric chromosomes. Centric fusions result in modifications to diploid number but do not influence the number of chromosome arms and are used in the karyotype evolution – not only in a large number of vertebrate species but also in insects and some plant species. Several examples are given. On the other hand, centric fusions, also called 'Robertsonian translocations', have severe clinical consequences in humans and economic importance in domestic animals. Detailed analysis of their causes and consequences is the substance of clinical cytogenetics.

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Mitosis and Meiosis Part A

Mariana C.C. Silva , ... Jan-Michael Peters , in Methods in Cell Biology, 2018

4.2 Chromosome Spreading and Giemsa Staining

Chromosome spreading combined with Giemsa staining is well suited for visualizing overall chromosome morphology. The harsh fixative, containing acetic acid, denatures proteins and is therefore often incompatible with antibody staining of proteins. However, the procedure is useful for analyzing chromosome segregation ( Waizenegger, Giménez-Abián, Wernic, & Peters, 2002), sister chromatid cohesion (Wirth et al., 2006), chromosome condensation (Giménez-Abián, Lane, & Clarke, 2018; Tedeschi et al., 2013), DNA double-strand breaks (Watrin & Peters, 2009), unresolved recombination intermediates, and chromosome fusions (Chan, Fugger, & West, 2018; Wechsler, Newman, & West, 2011). If interphase cells are spread, the morphology of their nuclei can also be visualized by this technique (Waizenegger et al., 2002).

1.

Fixed cells are spun down at 400   × g for 5   min and supernatant is removed up to 1   mL. Prior to spreading, the cell suspension is warmed up to RT.

2.

Spreading is done in an environment containing more than 40% humidity. 100   μL of cell suspension is dropped on slide from 0.5 to 1   m distance using P200 pipet.

3.

The slide with spread chromosomes is dried at RT for 1   h.

4.

Slide is put in a jar containing Giemsa solution for 7   min.

Note: Longer incubation might be necessary with reused Giemsa as well as removing oxidized Giemsa with Whatman filter paper before adding the slides.

5.

Slide is then submerged 2–3 times in a beaker containing tap water and then dried at RT.

6.

Slide can be mounted with Entellan and coverslip is added. Slides can be also used directly for microscopic detection (Fig. 6).

Note: Entellan should be completely dry before putting the slide under the microscope not to destroy objective.

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The In Vitro Chromosome Aberration Test

Marilyn Registre , Ray Proudlock , in Genetic Toxicology Testing, 2016

7.10.8 Slide Scoring

7.10.8.1 Basics

To produce reliable results, the slide reader should understand the basics of chromosome morphology and how aberrations are formed. Structural aberrations fall into two main categories:

•

breaks (including deletions) result from double-strand breaks (DSB) in the DNA that are not repaired

•

exchanges result from two or more DSBs with inappropriate rejoining/repair of the "sticky ends" within a single chromosome ("intrachanges") or between chromosomes ("interchanges")

The majority of clastogenic chemicals cause single-strand lesions that, during the course of DNA repair, can be converted to a DSB. If the DSB persist through the S phase, then it will be replicated and becomes evident as a chromatid break at metaphase [1]. In turn, if the chromatid break is not repaired, then it can be replicated, resulting in formation of a chromosome break. However, chromatid lesions are the major type of chemically induced damage seen using the standard sampling time of 1.5 cell cycles because they become evident at the first metaphase following induction [58]. Radiation and a few radiation-like chemicals can cause double-strand breakage directly; if this occurs after the S phase, then it will lead to chromatid lesions (breaks and exchanges); however, if it occurs prior to the S phase and is not repaired, then it will lead to chromosomal lesions involving both sister chromatids. Certain types of radiation-induced chromosome exchanges (notably dicentrics and centric rings) are accompanied by a paired fragment; in the case of chemicals, these derived aberrations are usually seen at the second metaphase after induction of damage and are not usually accompanied by fragments because acentric fragments tend to be lost during the first anaphase.

Gaps are achromatic lesions smaller than the width of one chromatid with minimum misalignment of the chromatid(s). Gaps are recorded but not scored as structural aberrations because they do not necessarily involve chromosome breakage. Note that identification criteria for gaps can vary between laboratories and countries. In addition, the width of the chromatids will depend on the degree of condensation, whereas not all chromatids have an equally clear outline. So, in a few cases, lesions classified as gaps may, in fact, be true breaks or even intrachanges.

Numerical aberrations (endoreduplication and polyploidy), if observed, are recorded and reported but are not necessarily indicative of genotoxicity. Apparent losses (in particular) or gains of one or two chromosomes in most metaphase figures are usually an artifact of slide preparation and therefore are not usually recorded or reported (i.e., the standard chromosome aberration test is not generally considered appropriate for detection of aneugenic events other than polyploidy).

Chemicals may have other observable effects on chromosomes (e.g., disruption of the centromere resulting in partial dissociation of the sister chromatids), which can be noted and reported as incidental observations. Although these may result in cytostatic or cytotoxic effects, they are not considered indicative of genotoxicity.

7.10.8.2 Understanding the normal karyotype

It is important to understand the structure of the individual chromosomes to facilitate identification of aberrations (e.g., chromosome breaks) and to avoid misclassification (e.g., secondary constrictions can appear similar to chromosome gaps); associations (attraction) of chromosomes via satellite bodies can give chromosome arrangements that appear similar to chromosome exchanges.

At metaphase, each pair of chromatids is joined by a constriction point called the centromere, which divides the chromosome into two arms. The short arm of the chromosome is termed the p (petite) arm and the longer arm is referred to as the q arm. The relative length of the chromosome and the ratio of the length of the p:q arms help identify the chromosome or the group of chromosomes.

•

Metacentric chromosomes have the centromere near the center of the chromosome.

•

Submetacentric means that the centromere is slightly off-center.

•

Acrocentric means that the centromere is distant from the center.

•

Telocentric means that the centromere is near one end of the chromosome.

The following descriptions refer primarily to diploid human metaphases (e.g., HPBL), but each laboratory should establish similar details (i.e., a chromosome map/karyotype) for any cell lines they use in the chromosome aberration test.

Diploid human cells contain 22 pairs of the autosomal chromosomes and a pair of sex chromosomes (XX for females and XY for males). Chromosomes are primarily identified and classified by size, with number 1 being the largest and chromosomes 22 and 21 being the smallest (they are approximately one-fourth the size of chromosome 1). The chromosomes are further classified into groups A to G:

•

Group A (1–3): Large metacentrics; individually identifiable by size and p/q ratio

•

Group B (4 and 5): Large submetacentrics

•

Group C (6–12 and X): Medium-sized metacentrics and submetacentrics

•

Group D (13–15): Medium-sized acrocentrics with satellites

•

Group E (16–18): Shorter metacentric/submetacentrics

•

Group F (19–20): Short metacentrics

•

Group G (21, 22, and Y): Short acrocentrics with satellites on 21 and 22.

Some chromosomes often have associated morphological characteristics:

•

Chromosomes 1, 9, and 16 secondary constrictions

•

D and G group chromosomes satellite associations

•

X and Y loss of centromeric activity

•

Y chromosome is often slightly distorted

The slide reader needs to aware of these features (particularly secondary constrictions and satellite associations) to avoid misidentification of aberrations.

7.10.8.3 Routine scoring

If microscopes are equipped with a vernier scale stage, then slides should be placed on the stage in a standard orientation each time as defined in the SOP (e.g., label to the left).

The coded slides should be methodically scanned at medium power (e.g., 16× objective). Metaphase spreads that are unbroken and show good morphology are subjected to detailed analysis. Under high power (100× oil-immersion objective), the chromosomes should be well defined and should not be in an early C-anaphase state with completely separated chromatids. To avoid the analysis of cells with random chromosome loss due to preparation artifacts, only cells with the modal (cell lines) or diploid number of centromeres ±2 in a single stage of condensation should be scored. During counting of the chromosomes, any structural aberrations or gaps should be recorded. The vernier reading of aberrant metaphases should be recorded against the aberration details. Especially during training, it can greatly facilitate review of results and correction of any misidentification if the slide reader draws a simple diagram for each recorded aberrant metaphase, indicating the approximate location within the cell of the lesion together with a stick drawing of the lesion.

Readable metaphases are identified by the following criteria:

•

chromosome/centromere number within ±2 of diploid (44–48 for HPBL) or modal number (for cell lines) in a single stage of condensation

•

well-spread with minimal overlap of chromosomes and chromosome arms

•

chromatids separate with centromere intact

•

structure of chromosomes clear and well-defined

If, for any reason, the required total of 150 readable metaphases is not obtained, then additional slides should be prepared and examined from the reserved fixed material from that culture. Sometimes, due to technical problems with slides from a particular culture, it may be necessary to read additional metaphases from the other duplicate culture to reach the desired total of 300 readable metaphases per experimental point. In that case, this should be mentioned and justified in the report.

7.10.8.4 Classification

Metaphases are classified into the three following categories.

1.

Normal metaphase (may have chromosome or chromatid gaps)

2.

Those with structural aberrations

3.

Numerical aberrations (polyploid, endoreduplicated, or hyperdiploid cells); these cells are not included in the readable metaphase totals

The main purpose of slide examination is to determine the proportion of metaphases with structural aberrations. A total of 300 readable metaphases (usually 150 per each of two cultures) per experimental point is examined for the presence of chromosome aberrations. Scoring less than this (e.g., when a clear positive response is evident) should be justified in the report and, ideally, the protocol.

The International System for Chromosome Aberration Nomenclature [59] is used to help classify structural aberrations into three main groups:

1.

Chromosome

a.

breaks/deletions

b.

exchanges

2.

Chromatid

a.

breaks/deletions

b.

exchanges

3.

Other (complex damage)

a.

multiple (multiple aberrations in a single cell, e.g., >5)

b.

pulverized chromosome

c.

pulverized metaphase where some or all the chromosome is pulverized

The chromatid and chromosome categories are divided into two subcategories: breaks (result of a single DNA break) and exchanges (result of two or more breaks with inappropriate rejoining). These subcategories can be further subdivided, but that does not seem to provide any useful additional information in the case of routine testing. Representative examples of lesions in human lymphocytes are shown in Figure 7.3.

Figure 7.3. Structural aberrations and other lesions in human lymphocytes.

Although much of the theory of how the various aberrations are formed was developed early during the development of the test, recent technical advances including the use of specific chromosome paints have helped confirm the details [1]. An excellent review of the mode of formation of chromosome lesions, their identification, and artifacts that might be misinterpreted as aberrations was presented by Natalie Danford in 2012 [58], whose company offers training and outsourced slide reading for cytogenetic toxicology tests (see http://www.microptic.com/).

Cytogenetic analysis has a subjective component, with even highly experienced analysts differing in their interpretation of the same cell. However, there are a number of instances in which misinterpretation can occur, most commonly resulting in a normal configuration being scored as aberrant, thus indicating the importance of recognizing secondary constrictions, satellites and satellite associations, and overlapping chromatids. There are also easily missed aberrations. Analysts need to be aware of these potential situations. Unfortunately, there is no substitute for experience.

Following is a list of potential misinterpretations analysts should be aware of:

Cause Confused With
Crossing-over of sister chromatids Dicentric
Satellite association: two chromosomes attracted by satellite regions Dicentric or exchange
Secondary constriction Dicentric
Secondary constriction Gap
Chromosomes overlapping near centromeres Exchange (quadriradial)
Chromosome twisting and overlapping Chromosome ring

Note that in the case of diploid cells, counting the centromeres can be a useful way of distinguishing artifacts from true lesions. Note that all these true lesions (with the exception of gaps) are relatively rare exchange events.

An example of a scoring sheet is illustrated in Figure 7.4. In this case, the standardized abbreviations for aberrations largely coincide with or are a simplification of those used by the ISCN.

Figure 7.4. Chromosome aberration scoring sheet example.

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Advances in Applied Microbiology

E.D. Garber , M. Ruddat , in Advances in Applied Microbiology, 2002

IV Cytology

An accurate count of the haploid chromosome number provides the expected number of linkage groups. Chromosome morphology can assign specific groups to specific chromosomes, and for favorable species, genes to a specific chromosome arm. The chromosomes of most fungal species, however, are notoriously small and difficult to count. Poon and Day (1974) could not get a precise count of the haploid chromosomes of M. violaceum, using phase contrast and fluorescence microscopy.

Pulse field gel electrophoresis (PFGE) provides an alternate method for separating fungal chromosomes (= bands in a gel). Perlin (1996) surveyed 18 strains of M. violaceum by PFGE and reported extensive chromosome polymorphism. The number of bands (= chromosomes) ranged from 6   + to 17–19. Based on genetic data, Day and Jones (1969) and Baird and Garber (1981) reported 14 linkage groups for the laboratory strains, indicating at least n  =   14 and an estimated n  =   20–21. Crosses between strains with the least and the most bands have not yet been made to determine teliospore viability and the number of bands in the viable meiotic products.

The origin of extensive chromosome polymorphism in fungal species is a problem yet to be resolved. A plausible explanation assumes that ectopic recombination, i.e., crossing over for homologous nucleotide sequences in different chromosomes, may be responsible. For M. violaceum, the Faust transposable element (see later discussion) presumably occurs in different chromosomes and could provide the proposed homologous nucleotide sequences (Garber and Ruddat, 1995). Families of transposable elements are found in the genomes of a number of fungal species (Daboussi, 1997).

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Conventional Genetic Manipulations

Rakesh Kumar Chahota , ... Shyam Kumar Sharma , in Lentils, 2019

4.3.1 Cytogenetic Manipulation

Chromosome analysis of lentils species revealed that the chromosomal numbers are stable in all the cultivated and wild species, and whatever variation found in the chromosome morphology is due to exposure of these species to climatic, biotic and abiotic stresses over a long period of time. A critical review of the literature revealed that all the lentil species, sub-species, cultivars, and germplasms of Lens contain 2n = 2   × = 14 chromosomes. There have been a number of cytological studies using various techniques in lentils, but there was no unanimity on chromosome morphology (metacentric, submetacentric, and acrocentric) karyotype formula, or total chromatin length (Bhattacharjee, 1951; Sharma and Mukhopadhyaya, 1963; Sinha and Acharia, 1972; Naithani and Sarbhoy, 1973; Gupta and Singh, 1981; Lavania and Lavania, 1983; Nandanwar and Narkhede, 1991). Ladizinsky (1993) used Feulgen staining and put all the controversy to rest by proposing that the Lens karyotype contains three types of chromosome; the first type is metacentric or submetacentric (centromere at or near the center). The second type is metacentric with a satellite, and the third type of chromosome is acrocentric (centromere is between the submetacentric and telomeric regions), and formulated with the 3   m/sm + 1   m with sat + 3   ac as a karyotypic formula. Balyan et al. (2002) also used modern techniques such as fluorescent in-situ hybridization (FISH) along with traditional Feulgen staining for chromosomal analysis of various Lens species, and observed that the position of secondary constriction in cultivated and some wild species, namely Lens culinaris, Lens orientalis, Lens odemensis, and Lens ervoides, was close to the centromere, that is, interstitial. However, in Lens nigricans, the position of secondary constriction was near the telomere of an acrocentric chromosome pair. Such cytological markers can be used to identify the hybrids as well introgression of desirable genes from Lens nigricans, but unfortunately, such methods have not been exploited to date in the lentil improvement programs. Application of fluorescent banding and FISH in the future may help to characterize apparently similar chromosomes at the constitutive heterochromatin level as an aid for future crop improvements.

Knowledge of cytological and molecular relationships between plant accessions is very useful in planning effective breeding strategies to transfer desirable genes or gene clusters from one species to another, thereby producing fruitful genomic reconstructions and disease free plants. Estimation of the genetic diversity of any crop species is a suitable precursor for improvement of the crop, because it generates baseline data to guide selection of parental lines and design of a breeding scheme. Genetic diversity refers to any variation in the nucleotides, genes, chromosomes, or genome of the organisms. Thus, each gene comprises a hereditary selection of DNA that occupies a specific place on the chromosomes and controls a particular characteristic of an organism (Welsh and McClelland, 1990).

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Small-Scale Gene Duplications

JOHN S. TAYLOR , JEROEN RAES , in The Evolution of the Genome, 2005

EARLY CHROMOSOMAL STUDIES

The science of genetics long predates the molecular definition of genes, and most early work in this area necessarily focused on observable changes in organismal phenotypes and chromosome morphology. Despite this obviously limited resolution, many of the key ideas surrounding duplications in genetic material were developed as part of these early investigations.

In 1911, for example, Kuwada proposed that the production of many different varieties of maize (Zea mays) was related to an ancient chromosome duplication event. At about the same time, Tischler (1915) noticed morphological differences that were correlated with variation in chromosome number in several closely related plants. This connection between chromosomal and morphological variation, though obvious today, was novel at the time. Notably, Tischler (1915) commented that "even recently, a drawing together of these two disciplines would have been considered absurd." The work of numerous scientists dealing with the importance of chromosome number, including that of Kuwada and Tischler (which was originally published in German), was reviewed by Winge in 1917. In his review, Winge remarked that chromosomes had by that time come to be regarded as being of quite extraordinary importance, the more so since the technical improvements in microscopy had made it possible to penetrate further into their nature. Indeed, many subsequent breakthroughs in the study of genomes, including recent ones, have been linked to comparable technological advances.

As far back as 1918, Calvin Bridges addressed the issue of how gene duplication might contribute to speciation and morphological innovation, as had been proposed by Kuwada and Tischler. Bridges remarked that the main interest in duplications lay in their offering a method for evolutionary increase in the lengths of chromosomes with identical genes, which could subsequently mutate separately and diversify in their effects (quoted in Bridges, 1935). Beginning in the 1920s and 1930s, this connection between gene duplication, speciation, and morphological innovation was addressed experimentally. Blakeslee (1934), for example, used Jimson weeds (Datura stramonium) to study the correlation between variation in karyotypes and organismal morphologies. Specifically, a diversity of lineages with unique karyotypes was observed in nature and created in the lab, and by comparing these strains, Blakeslee was able to associate morphological change with the duplication of specific chromosome parts. In one of his experiments, the duplication of a region of the largest chromosome caused the plant capsule to be small and the leaves to be narrow, whereas duplication of a different part of the same chromosome led to a large capsule and relatively broad leaves. Blakeslee made pure-breeding lab strains with duplicated chromosome segments and he considered these to be artificial new species. "Whether nature has used such methods, we do not yet know," he remarked, "but it should be remembered that often, when man has devised a method which he has thought unique, nature has been found to have had the priority in the use of the same method."

Hermann Muller, who, like Bridges, graduated from the famous Drosophila genetics lab of Thomas Morgan, produced fruit flies with a small fragment of their X chromosome duplicated and inserted into Chromosome 2 (Muller, 1935). Such duplications, thought Muller, might occur in nature and may be a way of increasing gene number without the typically negative consequences of gaining or losing one or more whole chromosomes. Muller (1935) further proposed that the redundant loci produced by the duplication of chromosome parts could experience divergent mutations and eventually be regarded as unrelated genes.

In 1936, Bridges was able to attribute phenotypic variation in Drosophila to the duplication of a particular locus when he concluded that the Bar and Bar-double phenotypes (both of which have reduced eyes) were a consequence of rare, small-scale tandem duplication events observable in giant, multistranded ("polytene") chromosomes (Figs. 5.1 and 5.2). These phenotypes had, in fact, been attributed to duplication events much earlier by Sturtevant (1925), but resolution was poor at that time because polytene chromosomes had not yet been discovered.

FIGURE 5.1. Polytene (multistranded) chromosomes in Drosophila, showing (A) the normal structure of regions 6 and 7 in the middle of the Y chromosome, (B) a heterozygote with a deletion of region 6F–7C in one of the chromosomes (indicated by the arrow), and (C) a reverse tandem duplication of region 6F–7C in the X chromosome.

 From Snustad et al. (1997), reproduced by permission (©John Wiley & Sons). Copyright © 1997

FIGURE 5.2. Effects of duplications for region 16A of the X chromosome on the size of the eyes in Drosophila. These phenotypic differences were attributed to duplication events by Sturtevant (1925), but polytene chromosomes were not known at that time, and it was Bridges (1936) who attributed these effects to the duplication of a particular locus.

 From Snustad et al. (1997), reproduced by permission (©John Wiley & Sons). Copyright © 1997

Serebrovsky (1938) not only recognized that selection could be relaxed in one of the genes in a duplicate pair, but also that both copies might be modified. Having characterized the roles of achaete and scute, closely linked genes on the Drosophila X chromosome that influence bristle morphology, Serebrovsky concluded that a single gene might influence multiple aspects of the phenotype and that after a duplication event, these multiple functions might be distributed between the duplicates. More specifically, he suggested that the loss of function of one duplicate should result in a specialization of genes, whereby each then fulfills only one function that is strictly limited and important for the life of the organism. This is a theme that will resurface later in the chapter.

Haldane (1932) also suggested that duplication events might be favorable because they produce genes that could be altered without disadvantage to the organism. In addition, he proposed that organisms with multiple copies of genes would be less prone to harmful mutations (Haldane, 1933). This suggestion was based upon Stadler's (1929) demonstration that polyploid species in Avena (oats) and Triticum (barley) were less sensitive to irradiation than their diploid congeners. The hypothesis that duplicate genes buffer organisms from harmful mutations continues to be tested in modern times (e.g., Wagner, 2000; Gu, 2003).

Goldschmidt (1940), Gulick (1944), and Metz (1947) were among the first to make explicit links between organismal complexity and gene duplication. Goldschmidt, who rejected the notion that organisms as different as humans and amoebae were connected by simple changes in the same set of genes, focused on the role of chromosome repatterning in macroevolution. Gulick argued that the whole history of many-celled organisms must undoubtedly have called for frequent increases in gene count as overall complexity increased, and Metz argued that evolution cannot be explained upon the basis of loss or simple alteration of materials already present in the germ-plasm. As Metz (1947) put it, "New elements must be added, otherwise we would have to assume that the primordial amoeba was endowed with all the germinal components now present throughout the wide range of its descendents, from protozoa to man." Using a style later adopted by Ohno, Metz (1947) argued that the duplication of chromosome parts, together with its consequences, has probably been one of the most important factors in evolution, if not the most important.

Twenty years before Ohno, but clearly expanding on ideas developed two decades earlier, Stephens (1951) doubted that evolution took place by the slow accumulation of small genic mutations. Recognizing that mutations were likely to impair original gene function, Stephens proposed that the only way of achieving "evolutionary progress" would be by increasing the number of genetic loci, either by the synthesis of new loci from nongenic material or by the duplication and subsequent differentiation of existing loci via genome duplication or unequal recombination (see later section). Also in 1951, Lewis concluded that numerous traits thought to be based on allele-level variation were actually under the control of closely linked duplicates, or so-called "pseudoalleles." Interestingly, Bailey et al. (2002) recently concluded that many single nucleotide polymorphisms (SNPs) in human genomes are, in fact, variants at duplicated loci—that is, pseudoalleles. Lewis (1951) also proposed a model for the creation of multistep biochemical reactions by gene duplication that presaged modern interpretations of duplicate gene evolution (see later section).

In summary, between 1911 and the 1950s, cytology and cytogenetics, with major contributions from Drosophila and plant research, produced much evidence for gene (and whole-genome) duplication events. Amazingly, all of this predated the elucidation of the double helix and the conclusive identification of DNA as the molecular basis of heredity. Moreover, many hypotheses concerning the mechanisms by which gene duplication might contribute to important evolutionary phenomena, including speciation and increases in morphological complexity, were developed during this early phase of gene duplication research.

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Molecular Diagnostics

Chand Khanna , Melissa C. Paoloni , in Withrow & MacEwen's Small Animal Clinical Oncology (Fourth Edition), 2007

Cytogenetics

Historically, these techniques involved the examination of metaphase preparations made from chromosomes. Metaphase preparations were then stained (banded) to help in the identification of distinct chromosome morphologies. Using these techniques, detection of gross abnormalities in chromosome number (ploidy) and the presence of chromosomal translocations were possible and led to the identification of genes associated with tumor development and progression. Cytogenetic analysis has been most useful in the clinical assessment of leukemias, where metaphase preparations are relatively easy to develop from whole blood samples. 6 For most human leukemias, cytogenetic descriptors are used to define distinct subgroups into prognostic groups and to guide treatment decisions. The use of cytogenetic approaches in the management of companion animals has been limited due to the difficulty in using conventional chromosomal banding to identify canine chromo somes. The development of chromosome-specific "paints" that allow the identification of specific canine chromosomes has improved the opportunity to apply cytogenetic descriptors to canine cancers. 7–12 Using these techniques, Breen and colleagues have identified a chromosomal translocation in many canine and feline cancers that includes a chromosomal translation syntenic for the bcr-abl Philadelphia chromosome in humans (personal communication, M. Breen). For the most part, traditional cytogenetic techniques, including the use of chromosome-specific paints, are labor intensive and have been replaced by alternative modalities. Comparative genomic hybridization (CGH) and, more recently, CGH arrays can define gains and losses in chromosome number within tumor specimens rapidly and with highly reproducible results. 8 A broader discussion of array-based diagnostics is provided in the section on RNA-based detection approaches, presented later in this chapter. CGH arrays are now routinely used to assess gains and loss for most human cancers. A first-generation CGH array has been developed for use in dogs. 11 Using this canine CGH array, a study has been undertaken into the chromosomal changes within a group of dogs with lymphoma (personal communication, K. Lindblad-Toh). Such genetic characterization of a diverse group of cancers, with distinct biological behaviors but similar histologic descriptions, will significantly improve opportunities to target specific therapies and management strategies to distinct biological subgroups of these diseases.

As specific abnormalities are defined in canine cancer, it is likely—as has been the case in human cancers—that simplified and often PCR-based (polymerase chain reaction; Figure 8-1) technologies will be adapted to rapidly identify these abnormalities as prospective diagnostic tests. 13–15 The use of PCR, using primer sets that bridge the translocation breakpoints, has largely replaced cytogenetic assessment of reciprocal translations seen in most translocation-positive cancers found in humans. Assessment of the size of a PCR product—and the opportunity to confirm the product identity by Southern blot or by sequence analysis—allows identification of not only the presence of a translocation but also the specific translocation. The presence of a translocation can also be verified using fluorescent in situ hybridization (FISH), with chromosome-specific paints. 8

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Molecular Diagnostics

Anne C. Avery , ... Melissa C. Paoloni , in Withrow and MacEwen's Small Animal Clinical Oncology (Fifth Edition), 2013

Detection of Chromosomal Abnormalities

Historically, these techniques involved the examination of metaphase preparations made from chromosomes. Metaphase preparations were then stained (banded) to help in the identification of distinct chromosome morphologies. Using these techniques, detection of gross abnormalities in chromosome number (ploidy) and of the presence of chromosomal translocations was possible and led to the identification of genes associated with tumor development and progression. Cytogenetic analysis has been most useful in the clinical assessment of leukemias, in which metaphase preparations are relatively easy to develop from whole blood samples. 6 For most human leukemias, cytogenetic descriptors are used to define distinct subgroups into prognostic groups and to guide treatment decisions. The use of cytogenetic approaches in the management of companion animals has been limited due to the difficulty in using conventional chromosomal banding to identify canine chromosomes. The development of chromosome-specific "paints" that allow the identification of specific canine chromosomes has improved the opportunity to apply cytogenetic descriptors to canine cancers. Using these techniques, Breen and colleagues have identified a chromosomal translocation in canine chronic myelogenous leukemia (CML) and chronic monocytic leukemia that is the equivalent of the bcr-abl Philadelphia chromosome found in human CML. 7,8

For the most part, traditional cytogenetic techniques, including the use of chromosome-specific paints, are labor intensive and have been replaced by alternative modalities. Comparative genomic hybridization (CGH) arrays can define gains and losses in chromosome number within tumor specimens rapidly and with highly reproducible results. In CGH analysis, the investigator labels genomic DNA from a normal individual and from tumor cells of the patient with two different color fluorescent probes. The labeled DNA is then hybridized to an array of DNA probes that span the majority of the genome. These probes are printed onto a chip or slide, such that the location of each individual probe is identified. The degree of hybridization to each probe is then determined by the level of fluorescence detected by laser excitation. Equal hybridization of the DNA from both sources to an individual probe indicates normal copy number, whereas increased binding by the tumor DNA indicates the presence of chromosomal duplication in the area of the genome covered by that probe (Figure 8-1). Similarly, higher binding by the DNA from the normal individual indicates chromosomal loss in the area.

CGH arrays are useful for localizing chromosomal regions where investigators should focus their search for genes important to that cancer. A canine CGH array with 2 megabase resolution has been reported. 9 Using this array, Breen and colleagues have shown that a subset of T-cell lymphomas (histologically defined as peripheral T-cell lymphoma, unspecified) exhibits copy number gain in regions common to most examples of this histologic type but not present in other T-cell lymphoma subtypes. 10 This finding will help to identify genes within the duplicated areas that might be useful for diagnostics and therapy and for understanding the genesis of the neoplasm. A similar study in canine malignant histiocytosis (MH) demonstrated that MH frequently exhibits loss of chromosomal regions that contain tumor suppressor genes. 11 Such genetic characterization of a diverse group of cancers, with distinct biologic behaviors but similar histologic descriptions, will significantly improve opportunities to target specific therapies and management strategies to distinct biologic subgroups of these diseases.

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GENETICS AND GENETIC RESOURCES | Cytogenetics of Forest Tree Species

Ž. Borzan , S.E. Schlarbaum , in Encyclopedia of Forest Sciences, 2004

Karyotype Analysis

The karyotype of an organism is a descriptive analysis of the chromosome complement. Each karyotype is defined numerically with statistical parameters of values based on the measurements of the chromosome's morphology. A graphic presentation is often used to give a better illustration of the chromosomes and their morphological features. Problems of comparison among studies can arise due to the lack of standardization in presenting a karyogram, graphically or numerically. An insight into this problem was published in Forest Genetics journal in 1996, with recommendations for standardized presentation of karyotypes for the species of the Pinaceae family. An example of the graphic presentation is shown in Figure 4.

Figure 4. Graphic presentation of karyotypes shown by the idiogram of Pinus nigra.

Classification of chromosomes by centromere position is a basic feature of karyotype analysis. Depending on the centromere position, chromosomes can range from metacentric to telocentric. Centromeric nomenclature, however, can vary from study to study. In studies of Pinus species, the classification presented by Saylor's classic papers is most often used. Another classification system often cited is the nomenclature presented by Schlarbaum and Tsuchiya in 1984, which was developed according to protocols given by Levan and his coworkers in 1964. Recognizing the inconsistency in centromeric nomenclature in a wide range of studies and the need for a standard, Levan and his coworkers developed precise standards for nomenclature and devised a system for modifying the standards to allow for better distinction among chromosomes if needed. Other modifications can be used if warranted by chromosome morphology, but the modifications should be according to their protocols.

The ability for rapid communication among scientists through the internet presents an exciting possibility in sharing karyomorphological data of investigated species. An idea for consolidating data in a standardized manner in a centralized database that can be instantly analyzed and made available worldwide via the internet was presented during the Second IUFRO Cytogenetics Working Party S2.04.08 Symposium, held in Graz, in 1998.

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Classical Cytogenetics

Marlys Houck , in Human Stem Cell Manual (Second Edition), 2012

Alternative Procedures

Other Species (Non-Human)

Harvest

Harvesting chromosomes from non-human species can generally be accomplished following the same procedures detailed above with minor alterations. It is important to determine the expected diploid number and chromosome morphology for the species before beginning. Species with high diploid numbers (mammals range from 2n = 6 to 106) may require a longer time in hypotonic solution to achieve sufficient chromosome spreading. Harvesting species with a high percentage of acrocentric (single-armed) chromosomes, such as mice may require reducing the strength and amount of time in colcemid to avoid producing overly short chromosomes with low resolution G-bands. Bi-armed chromosomes with the centromere at (metacentric) or near (submetacentric) the middle are usually not affected by colcemid as profoundly as acrocentric chromosomes.

Karyotyping

Information about the chromosome number, morphology and standard karyotype for a wide variety of species can be found individually in journal publications or in compilations such as the Atlas of Mammalian Chromosomes. We recommend examining the chromosomes initially by conventional Giemsa staining without banding. Numeric and morphologic abnormalities are easier to detect in non-banded preparations because the chromosomes are easier to count and the centromere is more visible than in banded chromosomes. After obtaining knowledge of the basic chromosome number and morphology, banding will allow positive identification of individual chromosomes and detection of rearrangements such as deletions and translocations.

Staining

These are two simple methods used to stain DNA, count chromosomes and observe centromere location, but they will not allow positive identification of individual chromosomes.

DAPI Staining
1.

Add a drop of mounting medium containing DAPI to the slide.

2.

Seal the coverslip.

3.

Count chromosomes under UV light using 100× oil immersion lens.

Conventional Giemsa Stain (Non-banded)
1.

Follow Steps 5–8 of procedure for GTG banding of chromosomes.

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