Source:  CURSO DE GENETICA CLINICA    Tag:  normal human karyotype


Cytogenetics is the study of chromosomes and their role in heredity.

Thus, this topic room is all about chromosomes: chromosome structure and composition, the methods that scientists use to analyze chromosomes, chromosome abnormalities associated with disease, the roles that chromosomes play in sex determination, and changes in chromosomes during evolution.
The field of cytogenetics emerged in the early twentieth century, when scientists realized that chromosomes are the physical carriers of genes. As is always the case in science, researchers built on the observations of their fellow investigators to synthesize the chromosome theory of heredity. This groundbreaking theory had its foundations in the detailed observations that cytologists had made about chromosome movements during mitosis and meiosis, which suggested that chromosome behavior could explain Mendel's principles of inheritance.
In the early years of cytogenetics, scientists had a difficult time distinguishing individual chromosomes, but over the years, they continued to refine the conditions for preserving and staining chromosomes to the reproducible standard that is now expected in clinical cytogenetics. (Looking back, it seems incredible that the human chromosome number was not established until 1955.) In today's procedures, metaphase chromosomes are treated with stains that generate distinctive banding patterns, and chromosome pairs are then arranged into a standardized format known as a karyotype. Among the members of a species, karyotypes are remarkably uniform, which has made it possible for cytogeneticists to detect various deviations in chromosome number and structure that are associated with disease states and developmental defects.
A normal human karyotype contains 22 pairs of autosomes and one pair of sex chromosomes. Aneuploidies, or changes in chromosome number, are easily detected on karyotypes. In humans, most aneuploidies are lethal because of the ensuing imbalance in gene expression. A notable exception is trisomy 21, or Down syndrome, which is frequently detected during prenatal screening of older mothers. Sex chromosome aneuploidies are also tolerated in humans, most likely because X inactivation maintains near-normal expression levels for X-linked genes. In addition to changes in chromosome number, karyotypes can also reveal more subtle changes in chromosome structure. In effect, the normal banding pattern of a chromosome provides a "bar code" that can be translated into a map of the chromosome. Cytogeneticists can then use coordinates on these rough chromosome maps, or idiograms, to identify the positions of structural abnormalities, including deletions, duplications, and translocations, to within a few megabases of DNA.
Over the past few decades, versatile methods based on fluorescence in situ hybridization (FISH) have transformed cytogenetics into a molecular science and provided cytogeneticists with powerful new tools. In FISH procedures, labeled DNA or RNA probes are hybridized with their complementary target DNA sequences on chromosomes. FISH experiments often generate colorful results, because multiple probes, each of which is labeled with a spectrally distinct fluorescent dye, can be used in the same experiment. The target DNA sequences may consist of either a single gene or a collection of genes spread out along the length of a chromosome. FISH procedures are now routinely employed in clinical cytogenetics. Spectral karyotyping provides an overview of any gross rearrangements and changes in chromosome number that have occurred in a patient's cells. Using gene-specific probes, cytogeneticists can also positively identify the genes affected by chromosomal mutations. More recently, researchers have additionally begun to employ comparative genomic hybridization to analyze small quantitative differences between individuals' DNA, including copy number variations (CNVs).
Outside the clinic, FISH is one of many techniques biologists use to investigate the structure of chromosomes and their organization within the nucleus. Although chromosomes may appear to be static structures when viewed under a microscope, cytogeneticists know that chromosomes are actually dynamic assemblies made up of a DNA-protein complex called chromatin. Chromatin undergoes dramatic changes in packing during the cell cycle, and its structure also varies locally along the length of each chromosome. Transcriptionally active chromatin, or euchromatin, has a different composition than silent chromatin, or heterochromatin. (The inactive X chromosome in female mammals is a special case in which heterochromatin extends along the entire length of a chromosome.) Some chromatin specializations are essential for normal chromosome behavior. For example, centromeres contain a unique chromatin that is required for chromosome attachment to the mitotic spindle. Likewise, chromosome integrity depends on the assembly of a specialized chromatin found exclusively at the telomeres. Other less defined aspects of chromosome structure may also be important in positioning individual chromosomes with the nucleus. For instance, mounting evidence seems to indicate that chromosomes occupy discrete territories in the interphase nucleus; this marks a significant departure from the previously accepted idea that chromosomes are randomly organized during interphase.
In this era of comparative genomics, cytogeneticis also offering insights into evolution. Using cross-species FISH, scientists have identified groups of genes, called synteny groups, that maintain the same linkage relationships with each other across species boundaries. Synteny data reveal numerous chromosomal rearrangements that have occurred during the course of evolution. Taken together with DNA sequence information, synteny data are proving useful for detecting genome duplications and for constructing phylogenetic trees.
The collection of articles in this topic room is intended to provide students with an introduction to chromosome biology and an appreciation of the experimental evidence that has led to the current state of understanding. Cytogenetics is a broad and growing field of research, and many topics have not been discussed in detail. The editors hope that this collection will grow over time as new discoveries are made and gaps in the current collection are filled. To this end, teachers and researchers are encouraged to contribute new articles to the collection after consultation with the editors.

Chromosome Territories: The Arrangement of Chromosomes in the Nucleus
By: Tom Misteli, Ph.D. (National Cancer Institute, Bethesda, MD) © 2008 Nature Education
Citation: Misteli, T. (2008) Chromosome territories: The arrangement of chromosomes in the nucleus. Nature Education 1(1)
..Chromosomes occupy specific regions of a nucleus, called "chromosome territories." These subdomains might be doing much more than just keeping everything organized.
1References and Recommended Reading.
Figure 1.Chances are that when most people hear the word "chromosome," they picture a rodlike, highly compacted chunk of DNA. While chromosomes do appear as condensed, elongated structures during the process of cell division, for most of a cell's lifetime, chromosomes do not look anything like this (Figure 1). In fact, some cell types, such as neurons, never undergo cell division, and their chromosomes therefore never take on a condensed state. So, what do chromosomes look like in the nucleus of a cell between cell divisions? Although this seems to be a simple question, its answer has only been revealed within the last two decades, and the implications of what has been found are both fascinating and the subject of much ongoing work in the field of genome cell biology.
Determining how chromosomes are organized inside the cellular nucleus was a technically challenging problem for scientists for many decades. One might think that it would be possible to simply use microscopes to peer into the nucleus and see how chromosomes are arranged. Indeed, during the 1960s and 1970s, various approaches involving both electron and light microscopy showed that chromosomes undergo a dramatic structural change as cells transition from mitosis to interphase, and that these structures exist as decondensed structures between cell divisions. However, microscopy did not reveal how chromosomes were organized in the nucleus of cells that were not undergoing mitosis. In fact, during the period between cell divisions, no distinct individual chromosomes can be recognized via either light or electron microscopy; thus, it is impossible to distinguish one chromosome from its neighbor using these methods.
Despite a lack of direct observational evidence, early researchers proposed two models for the way in which chromosomes were likely organized in the nuclei of nondividing cells. The first model, known as the chromosome territory model, was originally proposed by Carl Rabl in 1885. According to this model, the DNA of each chromosome occupies a defined volume of the nucleus and only overlaps with its immediate neighbors (Figure 2Ab). In contrast, according to the second or "spaghetti" model, the DNA fiber of multiple chromosomes meanders through the nucleus in a largely random fashion, and the chromosomes are therefore intermingled and entangled with each other (Figure 2Be).
Figure 2.The key experiment to distinguish between these two models was eventually carried out in the early 1980s by Thomas Cremer, a German cell biologist, and his physicist brother, Christoph Cremer. The Cremers realized that the two models made very distinct predictions and could thus be distinguished from each other if it were somehow possible to mark chromosome regions in part of the nucleus and follow the fate of these regions during cell division. Specifically, the Cremers argued that if chromosomes existed in territories occupying only a limited volume in the nucleus, marking of a defined volume would only affect very few immediately adjacent chromosomes (Figure 2Aa). On the other hand, if the spaghetti model were correct, the marking of a small volume would affect a large number of chromosomes, because fibers from many different chromosomes would be running through the marked volume (Figure 2Bd).
To carry out this decisive experiment, Christoph Cremer and his group developed a laser that could be focused very narrowly to shine on a small section of a cell's nucleus. The laser light was of a wavelength that induced DNA damage in the illuminated regions. The Cremers then exploited the ability of cells to repair damaged DNA by providing radioactively labeled nucleotides, which the cell incorporated into its DNA during the repair process. Then, when the cell entered the next mitosis and its chromosomes took on a condensed appearance, the marked regions were analyzed by radiography.
The results were clear. The Cremer brothers found that only a few chromosomes per cell were damaged, a result that strongly supported the chromosome territory model. The existence of chromosome territories was impressively confirmed a few years later by the development of the fluorescence in situ hybridization (FISH) technique, in which fluorescently labeled probes complementary to a specific chromosome are used to visualize a given chromosome in the intact nucleus. These experiments allowed direct visualization of the territorial nature of chromosomes (Figure 3).
Since its inception, the FISH technique has become one of the workhorse methods in the field of genome cell biology, and use of this technique has revealed many fundamental properties of chromosome territories. For instance, in combination with high-resolution microscopy, FISH has demonstrated that chromosome territories are irregular in shape but typically about 1 to 2 micrometers in diameter, and they consist of smaller subdomains. We now also know that chromosome territories exist in all higher eukaryotes, although chromosomes are more unfolded in lower eukaryotes such as yeast. Chromosome territories have additionally been found to border each other closely; in fact, neighboring chromosomes can invade each other's territories and intermingle at their peripheries. Observations of living cells have also revealed that chromosomes are essentially immobile, most likely held in place by the force exerted upon them by their neighbors. Furthermore, chromosome territories are semiconserved from parent to daughter cell during cell division, with locations in the daughter cell similar to those in the parent cell (Parada et al., 2003).
Figure 3: Visualization of chromosome territories by fluorescence in situ hybridization..A) Chromosome territories (green) in liver cell nuclei (blue). B) Visualization of multiple chromosomes reveals spatial patterns of organization. Chromosomes 12 (red), 14 (blue), and 15 (green) form a cluster in mouse lymphocytes.
Copyright 2004 BioMed Central Ltd., Parada, L. A., et. al., Tissue-specific spatial organization of genomes, Genome Biology 5, R44. Copyright 2002 Cell Press, Prada, L. A., et. al., Conservation of relative chromosome positioning in normal and cancer cells, Current Biology 12, 1692-1697
The fact that each chromosome occupies a distinct region of a cell's nucleus raises a fundamental question: Are chromosome territories randomly arranged in the nucleus, or they are they organized into patterns? Two experiments demonstrate that chromosomes are not randomly localized within the nucleus. First, when researchers measure the distance of a given chromosome from the center of a cell's nucleus, they note that some chromosomes localize toward the periphery, often touching the nuclear membrane, whereas others are located toward the center of the nucleus. Second, when visualizing multiple chromosome territories, scientists tend to see recurrent clusters of chromosomes. For example, in mouse lymphocytes, chromosome 12 often sits next to chromosome 14, which in turn is adjacent to chromosome 15, thereby forming a triplet cluster (Figure 3B).
Researchers have also examined whether various findings related to chromosomal territories hold true across species. Through such studies, scientists have noted that there are large areas of chromosomal identity between different species that have been maintained throughout evolution; moreover, these areas of identity maintain their positions in different species (Tanabe et al., 2002). Indeed, analysis of chromosome territories in many cell types and tissues has made it clear that patterns of chromosome arrangement are specific to both cell type and tissue type.
Yet another interesting observation has been the finding that chromosome territories can reposition in disease, which might provide novel insights into disease mechanisms and why genes are incorrectly expressed in disease. In fact, scientists have manipulated the localization of chromosomes and seen some changes in gene expression as a result, thus suggesting a possible mechanism for the connection between chromosomal territories and disease (Finlan et al., 2008).
Despite these discoveries, researchers still have numerous questions about chromosome territories. The holy grail of the field, for instance, is to understand what mechanisms determine exactly where a chromosome ends up in the nucleus. As of July 2008, no proteins have been identified that either anchor chromosomes in the nucleus or link multiple chromosomes to each other to establish chromosome clusters. As research continues, perhaps these and other questions will eventually be answered.