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During the Metaphase type of cell division in the Bone Marrow, Karyotyping method gives the visual representation of the 46 chromosomes paired and arranged in decreasing order of size. This representation is useful in leukemia purposes. This method is a difficult one because these chromosomes appear distorted, overlapped, and their images are usually blurred with undefined edges. So here, Karyotyping uses new mutual information method which is proposed to increase the discriminate power of the G-banding pattern dissimilarity between chromosomes and improve the performance of the classifier. This algorithm is formulated as such a method of combinatorial optimization problem. Where the distances between homologous chromosomes are minimized and the distances between non homologous ones are maximized. It is solved by using an integer programming approach. In this project chromosome dataset Lisbon-K1 (LK1) chromosome dataset with 9200 chromosomes was used for this study.
Introduction 1.1 The study of chromosome morphology and its relation with some genetic diseases is the main goal of cytogenetic. Normal human cells have 23 classes of large linear nuclear chromosomes, in a total of 46 chromosomes per cell. The chromosome contains approximately 30 000 genes (genotype) and large tracts of non coding sequences. The analysis of genetic material can involve the examination of specific chromosomal regions using DNA probes, e.g., fluorescent in situ hybridization (FISH) called molecular cytogenetic, comparative Genomic hybridization (CGH) , or the morphological and pattern analysis of entire chromosomes, the conventional cytogenetic, which is the focus of this paper. These cytogenetic studies are very important in the detection of acquired chromosomal abnormalities, such as translocations, duplications, inversions, deletions, monosomies, or trisomies. These techniques are particularly useful in the diagnosis of cancerous diseases and are the preferred ones in the characterization of the different types of leukemia, which is the motivation of this paper . The pairing of chromosomes is one of the main steps in conventional cytogenetic analysis where a correctly ordered karyogram is produced for diagnosis of genetic diseases based on the patient karyotype. The karyogram is an image representation of the stained human chromosomes with the widely used Giemsa Stain metaphase spread (G-banding) , where the chromosomes are arranged in 22 pairs of somatic homologous elements plus two sex-determinative chromosomes (XX for the female or XY for the male), displayed in decreasing order of size. A karyotype is the set of characteristics
extracted from the karyogram that may be used to detect chromosomal abnormalities. The metaphase is the step of the cellular division process where the chromosomes are in their most condensed state. This is the most appropriated moment to its visualization and abnormality recognition because the chromosomes appear well defined and clear. The pairing and karyotyping procedure, usually done manually by visual inspection, is time consuming and technically demanding. The application of the G-banding procedure to the chromosomes generates a distinct transverse banding pattern characteristic for each class, which is the most important feature for chromosome classification and pairing. The International System for Cytogenetic Nomenclature (ISCN) provides standard diagrams/ideograms of band profiles, as for all the chromosomes of a normal human, and the clinical staff is trained to pair and interpret each specific karyogram according to the ISCN information. Other features, related to the chromosome dimensions and shape, are also used to increase the discriminative power of the manual or automatic classifiers. 1.2 CHROMOSOME A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes,regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. Chromosomes vary widely between different organisms. The DNA molecule may be circular or linear, and can be composed of 100,000 to 10,000,000,000 nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without
which is tightly coiled in on itself. Chromosomal recombination plays a vital role in genetic diversity. for example. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. Chromosomes are the essential unit for cellular division and must be replicated. or it may unexpectedly evadeapoptosis leading to the progression of cancer. through processes known as chromosomal instability and translocation. Also. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). In practice "chromosome" is a rather loosely defined term. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. a large body of work uses the term chromosome regardless of chromatin content. the cell may undergo mitotic catastrophe and die. Unduplicated chromosomes are single linear strands. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. DNA is usually arranged as a circle. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. sometimes accompanied by one or more smaller. divided. In prokaryotes. In prokaryotes and viruses. This allows the very long DNA molecules to fit into the cell nucleus. Chromosomes may exist as either duplicated or unduplicated. If these structures are manipulated incorrectly. The structure of chromosomes and chromatin varies through the cell cycle. although there are many exceptions to this rule. In eukaryotes. However. the term genophore is more appropriate when no chromatin is present.defined nuclei) have smaller circular chromosomes. cells may contain more than one type of chromosome. circular DNA molecules called plasmids. These small circular genomes .
Chromosomal Mutations are substantial changes in chromosome structure that are large enough to be visible by karyotyping (see lab manual) and thus typically affect more than one gene. copies of chromosome 21. rather than 2.3 MUTATIONS IN CHROMOSOME NUMBER Normally. XX (female) or 46 XY (male). If the mutation involves only one or a few chromosomes in the genome (e. reflecting their bacterial origins. .XX. members of the same species have the same numbers of types of chromosomes (with the exception of sex chromosomes in males and females if sex is chromosomally determined).+21. Such individuals are called euploid and have the wild-type chromosome complement for the species. the individual carrying the mutation is said to be aneuploid. Euploid human karyotypes are 46. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. in which an individual has 3.+21. a extra copy of human chromosome 21).are also found in mitochondria and chloroplasts. The individual would have Down Syndrome and his/her karyotype would be written 47. An example of aneuploidy is trisomy 21.g.XY or 47. 1.
This is the only natural context in which individual chromosomes are visible with an optical microscope. the chromatids are uncoiled and DNA can again be transcribed. This compact form makes the individual chromosomes visible. . longer-lasting attachment in this region.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). and they form the classic four arm structure. During mitosis. A special DNA base sequence in the region of the kinetochores provides. 1. so that each daughter cell inherits one set of chromatids. q-g "grande").Fig 1. small) and the longer arms are called q arms (q follows p in the Latin alphabet. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. a pair of sister chromatids attached to each other at the centromere. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. one of which is present on each sister chromatid. The microtubules then pull the chromatids apart toward the centrosomes. Once the cells have divided. In spite of their appearance. along with special proteins. the chromatin strands become more and more condensed. chromosomes are structurally highly condensed. which enables these giant DNA structures to be contained within a cell nucleus. The shorter arms are called p arms (from the French petit. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II.
1. It is generally followed immediately by cytokinesis. bone marrow constitutes 4% of the total body mass of humans. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. which use the bone marrow vasculature as a conduit to the body's systemic circulation. bone marrow in large bones produces new blood cells. which divides the nuclei.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. . In humans.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones.6 kg (5. producing the lymphocytes that support the body's immune system CHAPTER 2 2. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells.  Bone marrow is also a key component of the lymphatic system. cytoplasm. in two separate nuclei. bone marrow accounts for approximately 2. On average. in adults weighing 65 kg (143 lbs).7 lbs).
genetically identical to each other and to their parent cell. which lack a nucleus. for instance during certain stages of fruit fly embryonic development. where the nuclear envelope breaks down before the chromosomes separate. metaphase. Prokaryotic cells. However. animals undergo an "open" mitosis. Mitosis occurs only in eukaryotic cells and the process varies in different species. to produce two identical daughter cells which are still diploid cells. For example. This accounts for approximately 10% of the cell cycle. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. forming single cells with multiple nuclei. Because cytokinesis usually occurs in conjunction with mitosis. . anaphase and telophase. cytokinesis and mitosis may occur independently. The process of mitosis is fast and highly complex. there are many cells where mitosis and cytokinesis occur separately. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. This occurs most notably among the fungi and slime moulds. but is found in various different groups. Even in animals. The cell then divides in cytokinesis. where chromosomes divide within an intact cell nucleus. prometaphase. These stages are interphase. The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. prophase. "mitosis" is often used interchangeably with "mitotic phase". divide by a process called binary fission.
The sister chromatids are held together by a specialized region of the chromosome known as the centromere. Each chromosome now has an identical copy of itself. These two cells are identical and do not differ in any way from the original parent cell. . Because each resultant daughter cell should be genetically identical to the parent cell. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. and together the two are called sister chromatids.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. the parent cell must make a copy of each chromosome before mitosis. This occurs during the S phase of interphase.
As a matter of convention. the parent cell will be split in half. pulling apart the sister chromatids of each chromosome. corresponding sister chromosomes are pulled toward opposite ends. . As the cell elongates. In animal cells. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. Eventually. Prokaryotic cells undergo a process similar to mitosis called binary fission. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. the process of binary fission is very much different from the process of mitosis. The chromosomes align themselves in a line spanning the cell. separating the two developing nuclei. each with a replica of the original genome. so they are renamed to sister chromosomes. A new nuclear envelope forms around the separated sister chromosomes. the cell pinches inward where the imaginary line used to be (the area of the cell membrane that pinches to form the two daughter cells is called the cleavage furrow). As mitosis completes. the daughter cells will construct a new dividing cell wall between each other. each sister chromatid is now considered a chromosome. However. the nuclear envelope which segregates the DNA from the cytoplasm disassembles.the cell begins cytokinesis.In most eukaryotes. In plant cells. giving rise to two daughter cells.
mainly via proteins. where the cell prepares itself for cell division. continues to grow as it duplicates its chromosomes (S). prophase is preceded by a pre-prophase stage. S (synthesis). grows more and prepares for mitosis (G 2).2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle. 2. In highly vacuolated plant cells. The phases follow one another in strict order and there are "checkpoints" that give the cell the cues to proceed from one phase to another. the cell grows by producing proteins and cytoplasmic organelles. During all three phases. However.1 Preprophase In plant cells only. the nucleus has to migrate into the center of the cell before mitosis can begin. and G2 (second gap).2. This is achieved through the formation of a phragmosome. a cell grows (G1). a transverse sheet of cytoplasm that bisects the cell along the future plane of cell .2. Thus. It alternates with the much longer interphase. chromosomes are replicated only during the S phase. Interphase is divided into three phases: G1 (first gap). and finally it divides (M) before restarting the cycle. All these phases in the interphase are highly regulated.
after the nuclear membrane breaks down. The cells of higher plants (such as the flowering plants) lack centrioles. preprophase is characterized by the formation of a ring of microtubules and actin filaments (called preprophase band) underneath the plasma membrane around the equatorial plane of the future mitotic spindle. These microtubules can attach to kinetochores or they can interact with opposing microtubules. aligned at the metaphase plate. This band marks the position where the cell will eventually divide. . microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. and microtubules have invaded the nuclear Prophase space. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. degraded. instead. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes.division. In addition to phragmosome formation. the pinched area is known as the cleavage furrow. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. The chromosomes have chromatin has condensed. Cytokinesis has already begun. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten.
Since the genetic material has already been duplicated earlier in S phase. A cell inherits a single centrosome at cell division. Close to the nucleus are structures called centrosomes. the genetic material in the nucleus is in a loosely bundled coil called chromatin. they are not essential for the . The centrosome is the coordinating center for the cell's microtubules. Chromosomes are typically visible at high magnification through a light microscope. giving a pair of centrosomes. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. which is replicated by the cell with the help of the nucleus before a new mitosis begins.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. bound together at the centromere by the cohesin protein complex. which are made of a pair of centrioles found in most eukaryotic animal cells. Although centrioles help organize microtubule assembly. chromatin condenses together into a highly ordered structure called a chromosome. At the onset of prophase. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. the replicated chromosomes have two sister chromatids.
it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. it is known that it contains some form of molecular motor. on an average 20 ). since they are absent from plants. This motor activity. provides the pulling force necessary to later separate the chromosome's two chromatids. the motor activates. and it occurs in most multicellular organisms.2. and centrosomes are not always used in mitosis. one attached at each chromatid. When the spindle grows to sufficient length. such as algae or trichomonads. 2. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle.formation of the spindle. . or its microtubules are able to penetrate an intact nuclear envelope. Although the kinetochore structure and function are not fully understood. kinetochore microtubules begin searching for kinetochores to attach to. Fungi and some protists.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. When a microtubule connects with the kinetochore. undergo a variation called closed mitosis where the spindle forms inside the nucleus. Each chromosome forms two kinetochores at the centromere. Prometaphase is sometimes considered part of prophase. This is called open mitosis. coupled with polymerisation and depolymerisation of microtubules. using energy from ATP to "crawl" up the tube toward the originating centrosome.
In the fishing pole analogy. in some sense. the chromosomes come under longitudinal tension from the two ends of the cell. . It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. In certain types of cells. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". only roughly lining up along the midline. analogous to a tug-of-war between people of equal strength. All chromosomes (blue) but one have arrived at the metaphase plate. the kinetochore would be the "hook" that catches a sister chromatid or "fish". This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. The centromeres of the chromosomes. an imaginary line that is equidistant from the two centrosome poles." Microtubules find and attach to kinetochores in prometaphase. As a result. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.3 Metaphase A cell in late metaphase. convene along the metaphase plate or equatorial plane. 2. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. Metaphase comes from the Greek meaning "after.
are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached.” “back. These two stages are sometimes called early and late anaphase. which have now become distinct sister chromosomes. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. The signal creates the mitotic spindle checkpoint.” “against. The force that causes the centrosomes to move towards the ends of the cell is still unknown.” or “re-”). At the end of anaphase.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). Two events then occur: first. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. These sister chromatids. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. the cell proceeds to anaphase (from the Greek meaning “up. Early anaphase is usually defined as the separation of the sister chromatids. allowing them to separate. Next. 2. . the proteins that bind sister chromatids together are cleaved. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. the nonkinetochore microtubules elongate.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate.
2. but rather a separate process. using fragments of the parent cell's nuclear membrane. cytokinesis is a separate process that begins at the same time as telophase. At telophase. unfold back into chromatin. cell . elongating the cell even more. It "cleans up" the after effects of mitosis. In both animal and plant cells. but cell division is not yet complete. now surrounded by new nuclei. Mitosis is complete. In animal cells. Corresponding sister chromosomes attach at opposite ends of the cell. the nonkinetochore microtubules continue to lengthen. Cytokinesis is technically not even a phase of mitosis. however. pinching off the separated nuclei. necessary for completing cell division. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. 2.5 Cytokinesis Cilliate undergoing cytokinesis. forms around each set of separated sister chromosomes. Both sets of chromosomes.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. A new nuclear envelope.
5. 2.5. separating the two nuclei. zygote and also the basis of the growth of a multicellular body.5. whereas some green algae use a phycoplast microtubule array during cytokinesis.. Similarly. Each daughter cell has a complete copy of the genome of its parent cell. New cells are formed by mitosis and so are exact copies of the cells being replaced. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. 2.division is also driven by vesicles derived from the Golgi apparatus. The end of cytokinesis marks the end of the M-phase. The phragmoplast is a microtubule structure typical for higher plants.e.2 Development and growth The number of cells within an organism increases by mitosis.4 Regeneration .3 Cell replacement In some parts of body. 2. This is the basis of the development of a multicellular body from a single cell i. Following are the occasions in the lives of organism where mitosis happens: 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. skin and digestive tract. cells are constantly sloughed off and replaced by new ones. e. which move along microtubules to the middle of the cell.1Significance Mitosis is important for the maintenance of the chromosomal set.g.5. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.
For example. and the latter cell having only one chromosome (the homologous chromosome).5. For example. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. Mitosis continues in the cells of bud and it grows into a new individual. a chromosome may fail to separate during anaphase.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. the hydra reproduces asexually by budding.7 Consequences of errors Although errors in mitosis are rare. The production of new cells is achieved by mitosis. resulting in binucleated cells. a condition known as monosomy. 2. In non-disjunction. a condition known as trisomy. Occasionally when cells experience nondisjunction. especially during early cellular divisions in the zygote. the process may go wrong. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). they fail to complete cell division and retain both nuclei in one cell. The cells at the surface of hydra undergo mitosis and form a mass called bud. These cells are considered aneuploid. 2. sea star regenerates its lost arm through mitosis. . One daughter cell will receive both sister chromosomes and the other will receive none. The same division happens during asexual reproduction or vegetative propagation in plants. a condition often associated with cancer.Some organisms can regenerate their parts of bodies.5.
sometimes mutuations occur in such genes and cells continue to divide. Now what happens is that cell abnormally continue to divide at a single place. its organelles disintegrate and reform in a matter of hours.Mitosis is a demanding process for the cell. As soon as they start to move and invade other cells there are said to be malignant tumours. Benign tumours are not harmful as soon as they are not moving. Occasionally. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. As long as these tumours remain in their original location they are called benign tumours. which goes through dramatic changes in ultrastructure. causing translocation. non-homologous chromosome. chromosomes may become damaged. It may reattach to the original chromosome. Errors in the control of mitosis may cause cancer. . It results in abnormal cell growth. causing chromosomal duplication. This phenomenon is called metastasis or spreading of disease. An arm of the chromosome may be broken and the fragment lost. The effect of these genetic abnormalities depends on the specific nature of the error. The fragment may incorrectly reattach to another. Or. and chromosomes are jostled constantly by probing microtubules. it results in the formation of Tumors. but in reverse orientation. causing deletion. it may be treated erroneously as a separate chromosome. Such tumours can send cancer cells to other parts in body where new tumours may form. causing inversion. All cells have genes that control the timing and number of mitosis. It results in the synthesis of execessive tissue growths. When tissues more than the requirement are synthesized in a single organ.
In certain types of cells. an imaginary line that is equidistant from the two centrosome poles.7 Metaphase Metaphase. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). Early events of metaphase can . chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. from the ancient Greek(between) and (stage). This process may also be referred to as endoreduplication and the cells as endoploid.2. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. only roughly lining up along the middleline. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. carrying genetic information. align in the middle of the cell before being separated into each of the two daughter cells. Metaphase accounts for approximately 4% of the cell cycle's duration. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. Preceded by events in prometaphase and followed by anaphase.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. resulting in cells with many copies of the same chromosome occupying a single nucleus. analogous to a tug of war between equally strong people. An example of a cell that goes through endomitosis is the megakaryocyte. 2.
Only after all chromosomes have become aligned at the metaphase plate. Chromosomes are condensed(Thickened) and highly coiled in metaphase. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. when every kinetochore is properly attached to a bundle of microtubules. securin. 2.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. as chromosomes with connected kinetochores will start the events of metaphase individually before other chromosomes with unconnected kinetochores that are still lingering in the events of prometaphase. Further they are used for slide preparation and banding (staining) of chromosomes to be visualised under microscope to study structure and number of chromosomes (karyotype). even if most of the kinetochores have been attached and most of the chromosomes have been aligned. Metaphase chromosomes make the classical picture of chromosomes (karyotype). produces a pattern of in total up to several hundred bands. does the cell enter anaphase. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor.coincide with the later events of prometaphase. Such a signal creates the mitotic spindle checkpoint. One of the cell cycle checkpoints occurs during prometaphase and metaphase. For classical cytogenetic analyses. often with Giemsa (G banding) or Quinacrine. which makes them most suitable for visual analysis. and separase. Staining of the slides. Normal metaphase spreads are used in . This would be accomplished by regulation of the anaphase-promoting complex.
. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. such as bcr-abl in chronic myelogenous leukemia. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. which may lead to chimeric oncogenes.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. for example. losses of chromosomal segments or translocations.
The study of whole sets of chromosomes is sometimes known as karyology. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. taxonomic relationships. and to gather information about past evolutionary events. .  The preparation and study of karyotypes is part of cytogenetics. The term is also used for the complete set of chromosomes in a species. Karyogram of human male using Giemsa staining. There may. and any other physical characteristics. So. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. Attention is paid to their length. and what they look like under a light microscope. cellular function. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). any differences between the sex chromosomes. in humans 2n = 46. or an individual organism. to study chromosomal aberrations. and the results may be used in evolutionary biology and medicine. banding pattern. be sex chromosomes. Thus.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. such as. Karyotypes can be used for many purposes. in normal diploid organisms. ordered by size and position of centromere for chromosomes of the same size. the position of the centromeres. autosomal chromosomes are present in two copies. or may not. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. The study of karyotypes is important for cell biology and genetics. Karyotypes describe the number of chromosomes.
Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. and he correctly insisted on humans having an XX/XY system. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912. Their behavior in animal (salamander) cells was described by Walther Flemming. The name was coined by another German anatomist. the discoverer of mitosis. The next stage took place after the development of genetics in the early 20th century. at first favoring 46. in 1882.3. von Waldeyer in 1888. which swells them and spreads the chromosomes . Considering their techniques. these results were quite remarkable. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. He revised his opinion later from 46 to 48. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. The subsequent history of the concept can be followed in the works of Darlington and White. Pretreating cells in a hypotonic solution. New techniques were needed to definitively solve the problem: 1. Using cells in culture 2. in contrast to their genic contents.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. concluding an XX/XO sex determination mechanism. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.
white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.2 Observations Six different characteristics of karyotypes are usually observed and compared: .2. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. For humans. a suitable dye.  Sometimes observations may be made on non-dividing (interphase) cells. such as Giemsa. Human chromosome 2 was formed by a merger of ancestral chromosomes. 3.2 Observations on karyotypes 3.2. reducing the number. is applied after cells have been arrested during cell division by a solution of colchicine. 3. the great apes have 48 chromosomes. Arresting mitosis in metaphase by a solution of colchicines 4.3. The sex of an unborn fetus can be determined by observation of interphase cells. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Usually.1 Staining The study of karyotypes is made possible by staining. Rather interestingly.
3. as well as other cytogenetic information. but the genes have been mostly translocated (added) to other chromosomes. and mainly consists of genetically inactive repetitive DNA sequences. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). Heterochromatin stains darker than euchromatin. Differences in absolute sizes of chromosomes.1. 6. indicating tighter packing. both have six pairs of chromosomes (n=6) yet V. permitting its loss without penalty to the organism (the dislocation hypothesis). Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. faba chromosomes are many times larger. 2. which (when they occur) are small bodies attached to a chromosome by a thin thread. Differences in degree and distribution of heterochromatic regions. Differences in the position of centromeres. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. . This is brought about by translocations. A full account of a karyotype may therefore include the number. Humans have one pair fewer chromosomes than the great apes. 4. This feature probably reflects different amounts of DNA duplication. 5. shape and banding of the chromosomes. type. Differences in number and position of satellites.
the same cannot be said for their karyotypes.3 The human karyotype Most (but not all) species have a standard karyotype.Variation is often found: 1. Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes. Between the germ-line and soma (between gametes and the rest of the body) 3. 3. which are highly variable. Any variation from the standard karyotype may lead to developmental abnormalities. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. males have both an X and a Y chromosome denoted 46. Normal karyotypes for females contain two X chromosomes and are denoted 46. and in . There is variation between species in chromosome number. Geographical variation between races 5. XX. Between the sexes 2. Between members of a population (chromosome polymorphism) 4. XY. Mosaics or otherwise abnormal individuals.
as in many sciarid flies. In some cases there is even significant variation within species. which were previously inexplicable. In A. despite their construction from the same macromolecules.. Chromosome elimination. the general significance of karyotype evolution is obscure.detailed organization. some organisms go in for large-scale elimination of heterochromatin. or other kinds of visible adjustment to the karyotype. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. 3.. entire chromosomes are eliminated during development. Chromatin diminution (founding father: Theodor Boveri). But. In this process. used in conjunction with other phylogenetic data.. portions of the chromosomes are cast away in particular cells.. despite many careful investigations. Godfrey and Masters conclude: "In our view. This variation provides the basis for a range of studies in evolutionary cytology.3. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. In a review. . "We have a very poor understanding of the causes of karyotype evolution. found in some copepods and roundworms such as Ascaris suum. In some species.1 Changes during development Instead of the usual gene repression. Although much is known about karyotypes at the descriptive level. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. it is quite unclear what the general significance might be.
where the haploid n = 1. they were astonished to find it had female = 6.suum. male = 7 chromosomes. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. was found to be 46. But when they obtained a couple more specimens they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the karyotype between (relatively) unrelated species is hugely variable. Muntiacus muntjak. Muntiacus reevesi.3. When they looked at the karyotype of the closely related Indian muntjac. The low record is held by the nematode Parascaris univalens. 3. They kept quiet for two or three years because they thought something was wrong with their tissue culture. all the somatic cell precursors undergo chromatin diminution. which was investigated by Kurt Benirschke and his colleague Doris Wurster.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac.. thus the mammalian female is a mosaic in respect of her X chromosomes. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). Xinactivation. the high record would be somewhere amongst the ferns.. "They simply could not believe what they saw. all telocentric. In placental mammals. The diploid number of the Chinese muntjac.. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. In marsupials it is always the paternal X which is inactivated. the inactivation is random as between the two Xs. The existence of supernumerary or B chromosomes .. In human females some 15% of somatic cells escape inactivation.
occurs mainly in plants. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants.means that chromosome number can vary even within one interbreeding population. 3.3 Fundamental number The fundamental number. It has been of major significance in plant evolution according to Stebbins.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. 15. Thus. Haplo-diploidy. Polyploidy. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. where one sex is diploid. and aneuploids are another example. but it has been significant in some groups. and in some other groups. FN. horsetails and psilotales) is also common.3. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. but in grasses the average is much higher. Humans have FN = 82. It is a common arrangement in the Hymenoptera. 21 and 22). Polyploidy in animals is much less common. where there are more than two sets of homologous chromosomes in the cells. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. and the other haploid.Endopolyploidy occurs when in adult differentiated . 14. 3. though in this case they would not be regarded as normal members of the population. Polyploidy in lower plants (ferns. FN ≤ 2n. about 70%. due to the presence of five acrocentric chromosome pairs (13.
5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. Down syndrome and Turner syndrome are examples of this. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. . but the nuclei contain more than the original somatic number of chromosomes. This process (especially studied in insects and some higher plants such as maize) may be a developmental strategy for increasing the productivity of tissues which are highly active in biosynthesis. See palaeopolyploidy for the investigation of ancient karyotype duplications. it is diverse and complex.tissues the cells have ceased to divide by mitosis. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). the daughter chromosomes separating from each other inside an intact nuclear membrane. and serves differentiation and morphogenesis in many ways. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. Abnormalities in chromosome number usually cause a defect in development. The cells do not always contain exact multiples (powers of two). which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. In many instances. 3. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication.
that the two chromosome morphs are adapted to different habitats. When this happens.Aneuploidy may also occur within a group of closely related species. Well-researched examples are the ladybird beetle Chilocorus stigma. 4.  Closer to home. some mantids of the genus Ameles. the European shrew Sorex araneus. and Crocus.000 km2). living from rainforests to . reducing the number. 6. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. In about 6. where the gametic (= haploid) numbers form the series x = 3.500 sq mi (17. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. the chromosome number is variable from one individual to another.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. where every number from x = 3 to x = 15 is represented by at least one species. 3.6 Species trees The detailed study of chromosome banding in insects with polytene chromosomes can reveal relationships between closely related species: the classic example is the study of chromosome banding in Hawaiian drosophilids by Hampton Carson. and 7. the great apes have 24x2 chromosomes whereas humans have 23x2. 5. 3. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. Classic examples in plants are the genus Crepis. Human chromosome 2 was formed by a merger of ancestral chromosomes.
at least into the Cretaceous. and skipping of islands. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. The results are clear. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain.4 million years ago (mya) (Mauna Kea) to 10mya (Necker).subalpine meadows. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. The inversions. gene arrangements are visible in the banding patterns of each chromosome. probably 20 million years ago. in the family Drosophilidae. which can be dated to 30 mya. but these are much less frequent. make it possible to see which species are closely related. especially inversions. . Using K-Ar dating. when plotted in tree form (and independent of all other information). Chromosome rearrangements. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. Drosophila and Scaptomyza. There are also cases of colonization back to older islands. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. show a clear "flow" of species from older to newer islands. In a sense. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. it is more likely to have been a group from the same species. the best-studied group of Hawaiian drosophilids. the present islands date from 0. The polytene banding of the 'picture wing' group. Although it would be possible for a single gravid female to colonise an island. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll.
the dark regions tend to be heterochromatic.7. It yields a series of lightly and darkly stained bands .There are other animals and plants on the Hawaiian archipelago which have undergone similar. if less spectacular. • T-banding: visualize telomeres. The pattern of bands is very similar to that seen in G-banding.7 Depiction of karyotypes 3. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). adaptive radiations. R-banding is the reverse of G-banding (the R stands for "reverse"). The light regions tend to be euchromatic. • • C-banding: Giemsa binds to constitutive heterochromatin.1 Types of banding Cytogenetics employs several techniques to visualize different aspects of chromosomes: • G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. early-replicating and GC rich. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. human genome. . This method will normally produce 300-400 bands in a normal. so it stains centromeres. late-replicating and AT rich. 3.
Quinacrine binds to the adeninethymine-rich regions. This yields a dark region where the silver is deposited. denoting the activity of rRNA genes within the NOR. less frequently Quinacrine. is used to stain bands on the chromosomes. a dye. and the long arm on the bottom. often Giemsa (G-banding). Giemsa is specific for the phosphate groups of DNA. respectively. Cri du chat syndrome involves a deletion on the short arm of .2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. In addition. 3. Karyotypes are arranged with the short arm of the chromosome on top. Some karyotypes call the short and long arms p and q.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. both chromosomes in a pair will have the same banding pattern. For example. In the "classic" (depicted) karyotype.7. Each chromosome has a characteristic banding pattern that helps to identify them.
. The critical region for this syndrome is deletion of 15.2) 3. Image processing software then assigns a pseudo color to each spectrally different combination.del(5)(p15. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.XX.chromosome 5. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.2.3 Spectral karyotype (SKY technique) Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. 3. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. allowing the visualization of the individually colored chromosomes. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough.5p-. This method is also known as virtual karyotyping. It is written as 46. a combinatorial labeling method is used to generate many different colors.7.XX. which is written as 46. Because there are a limited number of spectrally-distinct fluorophores.
CHAPTER 4 .
is caused by trisomy of chromosome 21. also known as aneuploidy. Down syndrome. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. X0).4. large-scale deletions or duplications.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. inversions. although they generally do not survive to birth. as in derivative chromosome. Structural abnormalities often arise from errors in homologous recombination. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. as in the presence of extra or missing chromosomes. often occur as a result of nondisjunction during meiosis in the formation of a gamete. otherwise known as 47. including . trisomies. a common chromosomal disease. translocations. trisomy 9 and trisomy 16. Numerical abnormalities. Some disorders arise from loss of just a piece of one chromosome. the most common male chromosomal disease. Also documented are trisomy 8. are common numerical abnormalities. in which three copies of a chromosome are present instead of the usual two. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. XXY is caused by an extra X chromosome. or structural. Klinefelter syndrome. X or 45. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. • • Patau syndrome is caused by trisomy of chromosome 13.
example of imprinting disorder. from the loss of part of the short arm of chromosome 1. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. a deletion of the paternal genes. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. A chromosome anomaly. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. caused by abnormal formation of the larynx. • Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing.• Cri du chat (cry of the cat). Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. The name comes from the babies' distinctive cry. one well-documented example is the Philadelphia chromosome. example of imprinting disorder. numerical and structural anomalies. A Karyotype refers to a full set of chromosomes from an individual which can be compared to a "normal" Karyotype for the species via genetic testing. A chromosome anomaly may be detected or confirmed in this manner. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia. from a truncated short arm on chromosome 5. They can be organized into two basic groups. a deletion of the maternal genes. . 1p36 Deletion syndrome. There are many types of chromosome anomalies.
Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. rather than two). also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21.). This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. and Jacobsen syndrome. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. an X.3 Structural abnormalities When the chromosome's structure is altered. There are two main types of translocations. also called the terminal 11q deletion disorder. Tetrasomy. Known human disorders include Charcot-Marie-Tooth disease type 1A which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. • • Translocations: When a portion of one chromosome is transferred to another chromosome. segments from two different chromosomes have been exchanged.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). In humans an example of a condition caused by a numerical anomaly is Down Syndrome. Known disorders in humans include Wolf-Hirschhorn syndrome. 4. etc. resulting in extra genetic material. Duplications: A portion of the chromosome is duplicated. In a reciprocal translocation. which is caused by partial deletion of the short arm of chromosome 4. an entire chromosome has . In a Robertsonian translocation.4.
• Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. especially the chromosomes. the anomaly is present in every cell of the body. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. 4.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. This can happen with or without loss of genetic material. however.in humans these only occur with chromosomes 13. resulting in Mosaicism (where some cells have the anomaly and some do not). and are therefore initially not inherited. Chromosome anomalies can be inherited from a parent or be "de novo". turned upside down and reattached. It includes routine analysis of G-Banded chromosomes. • Rings: A portion of a chromosome has broken off and formed a circle or ring. Some anomalies. therefore the genetic material is inverted. 4.attached to another at the Centromere . They often lead to an increased tendency to develop certain types of malignancies. 14. as well . Therefore. other cytogenetic banding techniques.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. 21 and 22. • Inversions: A portion of the chromosome has broken off. 15. can happen after conception. This is why chromosome studies are often performed on parents when a child is found to have an anomaly.
Their behavior in animal (salamander) cells was described by Walther Flemming. New techniques were needed to definitively solve the problem: 1. Considering their techniques. He revised his opinion later from 46 to 48. the discoverer of mitosis. concluding an XX/XO sex determination mechanism. which swells them and spreads the chromosomes . The name was coined by another German anatomist. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Using cells in culture 2. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. at first favoring 46. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Pre-treating cells in a hypotonic solution. von Waldeyer in 1888. 4. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. these results were quite remarkable. in 1882.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). The next stage took place after the development of genetics in the early 20th century. Investigation into the human karyotype took many years to settle the most basic question: how many chromosomes does a normal diploid human cell contain? In 1912.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. in contrast to their genic contents. and he correctly insisted on man having an XX/XY system.
persimilis from wild populations in California and neighboring states. Human chromosome 2 was formed by a merger of ancestral chromosomes. 4. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. reducing the number.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Using Painter's technique they studied the polytene . 4.3. During her cytogenetic work. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Squashing the preparation on the slide forcing the chromosomes into a single plane 5. In 1931. Rather interestingly.6. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. a find which eventually led to her Nobel Prize in 1983. Arresting mitosis in metaphase by a solution of colchicine 4. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. McClintock discovered transposons.2 Natural populations of Drosophila In the 1930s.6. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.6 Applications in biology 4. the great apes have 48 chromosomes.
Down syndrome is also referred to as trisomy 21. discoveries were quickly made related to aberrant chromosomes or chromosome number. 4. as they would if selectively neutral. In some congenital disorders. Evidence rapidly accumulated to show that natural selection was responsible. but adjust to certain frequencies at which they become stabilised. It was found that the various chromosome types do not fluctuate at random. breeding and sampling whilst preventing escape. This had the benefit of eliminating migration as a possible explanation of the results.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. which enabled feeding. as with most polymorphisms. By the time Dobzhansky published the third edition of his book in 1951 he was persuaded that the chromosome morphs were being maintained in the population by the selective advantage of the heterozygotes. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. Dobzhansky bred populations in population cages. In 1959. such as Down's syndrome. Abnormalities arising from nondisjunction events can cause cells with aneuploidy (additions or deletions of entire chromosomes) in one of the parents or in the fetus. Using a method invented by L'Heretier and Teissier. .
and XXXX. resulting in 47 total chromosomes. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. is used today as a diagnostic for CML. Pennsylvania. Many other sex chromosome combinations are compatible with live birth including XXX.as both scientists were doing their research in Philadelphia. In 1960. with the development of more advanced techniques. has Klinefelter's Syndrome. Thirteen years later. This abnormal chromosome was dubbed the Philadelphia chromosome .Other numerical abnormalities discovered include sex chromosome abnormalities. an additional X chromosome in a male. in addition to other tests. which is required in normal females to compensate for having two copies of the chromosome. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. Identification of the Philadelphia chromosome by cytogenetics. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. . An individual with only one sex chromosome (the X) has Turner syndrome. XYY. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). Not all genes on the X Chromosome are inactivated.
Caspersson developed banding techniques which differentially stain chromosomes. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material.FIG Advent of banding techniques In the late 1960s.8 Beginnings of molecular cytogenetics . and elongation techniques for all culture types that allow for higher resolution banding. Deletions within one chromosome could also now be more specifically named and understood. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 4. These maps became the basis for both prenatal and oncological fields to quickly move cytogenetics into the clinical lab where karyotyping allowed scientists to look for chromosomal alterations. Deletion syndromes such as DiGeorge syndrome.
1 Karyotyping . This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). While radioisotope-labeled probes had been hybridized with DNA since 1969. cloned and studied in ever greater detail.In the 1980s. advances were made in molecular cytogenetics. Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. movement was now made in using fluorescent labeled probes. CHAPTER 5 Techniques 5.
Routine chromosome analysis (Karyotyping) refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa, Leishmanns, or a mixture of the two. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing. Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce specific banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding (Rbanding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q-bands. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nucleolar organizing region stains (NOR stains). These latter methods specifically stain certain portions of the chromosome. C-banding stains the constitutive heterochromatin, which usually lies near the centromere, and NOR staining highlights the satellites and stalks of acrocentric chromosomes. High-resolution banding involves the staining of chromosomes during prophase or early metaphase (prometaphase), before they reach maximal condensation. Because prophase and prometaphase chromosomes are more extended than metaphase chromosomes, the number of bands observable for all chromosomes increases from about 300 to 450 to as many as 800. This allows the detection of less obvious abnormalities usually not seen with conventional banding.
5.2 Slide preparation Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, umbilical cord, chorionic villi, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor are removed, and replaced with a hypotonic solution. This causes the white blood cells or fibroblasts to swell so that the chromosomes will spread when added to a slide as well as lyses the red blood cells. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis. 5.3 Analysis Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009). 5.4 Fluorescent in situ hybridization
Fluorescent in situ hybridization refers to using fluorescently labeled probe to hybridize to cytogenetic cell preparations. In addition to standard preparations FISH can also be performed on:
• • • • • • •
bone marrow smears blood smears paraffin embedded tissue preparations enzymatically dissociated tissue samples uncultured bone marrow uncultured amniocytes cytospin preparations
5.5 Slide preparation The slide is aged using a salt solution usually consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are then co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2phenylindole (DAPI) or propidium iodide. 5.7 Analysis Analysis of FISH specimens is done by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease,
including signal and image processing. such as C. Using MATLAB. CGH and Single nucleotide polymorphism-arrays. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. data analysis. C++. control design. . data visualization. you can solve technical computing problems faster than with traditional programming languages. You can use MATLAB in a wide range of applications. Future of cytogenetics Advances now focus on molecular cytogenetics including automated systems for counting the results of standard FISH preparations and techniques for virtual karyotyping. For congenital problems usually 20 metaphase cells are scored. and FORTRAN.generally between 200 and 1000 cells are counted and scored. and computational biology. such as comparative genomic hybridization arrays. communications. and numerical computation. financial modeling and analysis. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. test and measurement.
you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. With the MATLAB language.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. and distribute your MATLAB algorithms and applications. MATLAB eliminates the need for ‘for’ loops. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. The image brightness and contrast depend on the specific tuning of the microscope and the particular geometric shape of each chromosome depends on the specific metaphase plaque from which the chromosomes were extracted. such as declaring variables.MATLAB provides a number of features for documenting and sharing your work. It enables fast development and execution. You can integrate your MATLAB code with other languages and applications. In many cases. . and allocating memory. specifying data types. 6. one line of MATLAB code can often replace several lines of C or C++ code. These effects must be compensated to improve the results of the pairing algorithm. The image processing step is composed of the following operations. As a result.
To compare chromosomes from a band pattern point of view.2 Concepts used in this phase 1) Image conversion 2) Denoising .1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. Therefore. To compensate for this inhomogeneity. a dimensional scaling is performed before the pattern features is extracted to make all the chromosome with the same size and aspect ratio by interpolating the original images. or at least attenuated. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. 2) Geometrical compensation—The geometric compensation. 6. geometrical and dimensional differences must be removed. This compensation algorithm is composed of the following main steps: a) chromosome and medial axis segmentation b) axis smoothing c) interpolationalong orthogonal lines to the smoothed medial axis d) border regularization 3) Shape normalization—The features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding). the spatially scaled images are histogram equalized.
For example.I.I. For example. For example. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension.I). green. In addition to these image type conversion functions. MATLAB simply applies the filter to the indices in the indexed image matrix. there are other functions that return a different image type as part of the operation they perform. if you want to filter a color image that is stored as an indexed image. RGB = cat (3.2. If you attempt to filter the indexed image. MATLAB filters the intensity values in the image. . you must first convert it to true color format. and blue planes. and the results might not be meaningful. When you apply the filter to the true color image. You can perform certain conversions just using MATLAB syntax. 6. The resulting true color image has identical matrices for the red. as is appropriate. listed in the following table. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. so the image displays as shades of gray.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another.3) Edge detection 4) Two dimensional convolutions.
There is a large number of edge finding algorithms in existence. . to isolate particular objects from their background. to recognize or classify objects. and hence the type of noise on the image. we may expect errors to occur in the image signal.5 Edge detection Edges contain some of the most useful information in an image.parameters. and we shall look at some of the more straightforward of them. If an image is being sent electronically from one place to another. caused by external disturbance. ) Where the parameters available depend on the method used 6. If one of these matrices describes a two-dimensional finite impulse response . hence we can choose the most appropriate method for reducing the effects.3 Two dimensional convolutions C = conv2(A. via satellite or wireless transmission.4 Denoising We may define noise to be any degradation in the image signal. We may use edges to measure the size of objects in an image. The general Matlab command for finding edges is edge(image.2.6. Usually we know what type of errors to expect. Cleaning an image corrupted by noise is thus an important area of image restoration.'method'. or through networked cable. .2.B) computes the two-dimensional convolution of matrices A and B. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. 6.
7).bmp'). edge(im1. If hcol is a column vector and hrow is a row vector.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. this case is the same as C = conv2(hcol*hrow.na] and the size of B is [mb.0.hrow.'shape') subsection of the two-dimensional convolution. minus one.A).'sobel').nb].na+nb-1]. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. nb]+1)/2).. if the size of then the size of C is [ma+mb-1.. The indices of the center element of B are defined as floor(([mb C = conv2(hcol. The size of matrices. . rgb2gray im2bw(im.. That is.(FIR) filter. imedfilt2(im1. the other matrix is filtered in two dimensions. as specified by the shape parameter Algorithms description 1) Read the image and convert into gray 2)Remove noise 3) Background separation 4) Edge detect 5) Separate the pairs MODULE 1 PSEUDO CODE iimread('12345.[3 3]). C = conv2(.
j)==L_number(k) flag=1.n]=size(L). L_number=zeros(mx. Index=1.1).1). Msk conv2(double(BW). bwlabel(B. MODULE 2 clc [m. rc = [r c].c] = find(L==22).imy]=size(BW). flag=0. for i=1:sx x1=rc(i. [r. y1=rc(i.2). .imy).y1)=255.8).double(msk)). [sx sy]=size(rc). for i=1:m for j=1:n if L(i. nzeros(imx.[imx.j)~=0 for k=1:mx if L(i. n1(x1. mx=max(max(L)).
45. n1(x220.127.116.11). for i=1:sx x1=rc(i.18.104.22.168.19.9.48. rc = [r c].4. end end L_number.14.imy). Index=Index+1.21.).32. y1=rc(i.22.214.171.124. end.2).40.j).c] = find(L==L_number((Test_number(x)))).57.38. .43.20. 126.96.36.199.62. [sx sy]=size(rc).33.30. end flag=0. end %h=figure.66].imshow(n1.39. n1=zeros(imx. for x=1:46 [r.end end if flag~=1 L_number(Index)=L(i.188.8.131.52.22.52.24.y1)=255.41.65.10.26. Test_number=[3.50.6.
s=bwmorph(skel. end end end Circumference(i)=Circumference_sum. Circumference_sum=0.1).y)==1 Circumference_sum=Circumference_sum+1. .'. for x=1:m for y=1:n if BW1(x.1. skel=im2bw(skel.'canny'). BW1=edge(BW. s1=bwmorph(s.'skel'.end Circumference=zeros(46. f=imcomplement(f). BW=im2bw(f). Arm_length=zeros(46. [m n]=size(BW1).1).8).Inf).1). for i=1:46 f=imread(strcat(num2str(i).5*graythresh(skel)). BW=double(BW).bmp')). Arm_length_sum=0. skel=im2double(f).'spur'. Area=zeros(46.
end end end Area(i)=Area_sum. Area_sum=0. for x=1:m for y=1:n if BW(x.[m n]=size(s1). Arm_length.y)==1 Area_sum=Area_sum+1. . [m n]=size(BW). for x=1:m for y=1:n if s1(x.y)==1 Arm_length_sum=Arm_length_sum+1. end Circumference. end end end Arm_length(i)=Arm_length_sum. BW=im2bw(f).
2)=i+1.2)==46 Pair(46.1)=46.1)=i.Area.2)=i. Pair(i.2). end end Pair.1)=i.2)=j. Pair(i. Pair(46. Pair(i. for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)). for i=1:45 min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)Arm_length(i+1))+abs(Area(i)-Area(i+1)). . end end end for i=1:45 if Pair(i. Pair(i. Pair=zeros(46.
end end if flag~=1 if figure_flag~=47 subplot(23. end f1=imread(strcat(num2str(Pair(i.2)).bmp')). delete(figure_flag)=Pair(i. if figure_flag~=47 subplot(23.1)==delete(j) flag=1. end flag=0. figure_flag=figure_flag+1.delete=zeros(46.1). end f2=imread(strcat(num2str(Pair(i.figure_flag). figure_flag=figure_flag+1. for i=1:46 for j=1:46 if Pair(i.'. flag=0.1)).'. figure_flag=1.figure_flag).2. .2. imshow(f1).2). imshow(f2).bmp')).
based on the MI. Copenhagen. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. and Philadelphia. in the scope of karyotyping process used in cytogentic analysis. The images of these chromosomes present less quality and level of detail than the ones usually used in traditional genetic analysis using datasets such as Edinburgh.end CONCLUTION In this paper. dimensions and banding profiles. The proposed algorithm is based on the traditional features extracted from the karyogram. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. The main goal of this paper is to provide useful contributions toward the design of a fully automatic chromosomes pairing algorithm of bone marrow cells to be used in the diagnosis of leukemia. 2) feature extraction from the processed images . such as. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. plus a new one.
Vectors of coefficients associated with each one of the 22 classes are computed and the distance between two arbitrary chromosomes is the minimum one among all distances obtained with these 22 vectors. Tests using 19 karyograms based on bone marrow cells. Here. extracted from the unordered karyogram. achieves a 70. and band pattern. This normalization is needed to make it possible the band pattern comparison between chromosomes. shape and band pattern. and finally. The training process consists in the estimation of each vector of coefficient . The addition of the MI feature to the traditional geometrical and band profile features described in the literature leads to a clear improvement in the . a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature.working within an 8-D feature space. 4) pairing. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. by minimizing the overall distances between chromosomes of the same class (intraclass) and by maximizing the distances between chromosomes when at least one of them does not belong to that class (interclass). and to normalize their dimensions. the romosome images. In the image processing step.characterizing the size.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. The features extracted from the processed images discriminate each pair with respect to their size. shape. The pairing process is performed by efficiently solving a combinatorial problem where a permutation matrix is obtained from the distance matrix computed with the extracted features associated with each pair of chromosomes in the karyogram. from the chromosomes in the training set. are processed in order to compensate for geometrical and intensity distortions. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts.10% mean classification rate.
g. This dataset was made publicly available .10% classification ratewas obtained. e. called LK1 . despite the low quality of this type of chromosomes. amean classification rate larger than 93% was obtained in all experiments. whose images are of significantly higher quality.performance of the classifier. Copenhagen. Using 27 karyograms andworking with a limited number of classes (≤ 8).. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. In addition. Executing the algorithm on a higher quality dataset. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. centromere position. a 76. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. presenting a uniform level of condensation. REFERENCES . The results presented in this paper are promising. or Philadelphia. In fact. such as Edinburgh. and from which it is possible to extract additional features. a new chromosome dataset with 9200 chromosomes from bone marrow cells.
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