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[1] nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without

Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). These small circular genomes . The structure of chromosomes and chromatin varies through the cell cycle. Unduplicated chromosomes are single linear strands. circular DNA molecules called plasmids. the cell may undergo mitotic catastrophe and die. a large body of work uses the term chromosome regardless of chromatin content. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. However. or it may unexpectedly evadeapoptosis leading to the progression of cancer. divided. which is tightly coiled in on itself. through processes known as chromosomal instability and translocation. the term genophore is more appropriate when no chromatin is present. In eukaryotes. Chromosomes are the essential unit for cellular division and must be replicated. Chromosomes may exist as either duplicated or unduplicated. for example. cells may contain more than one type of chromosome. DNA is usually arranged as a circle. although there are many exceptions to this rule. In practice "chromosome" is a rather loosely defined term. This allows the very long DNA molecules to fit into the cell nucleus. If these structures are manipulated incorrectly. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. sometimes accompanied by one or more smaller. In prokaryotes. Also. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny.defined nuclei) have smaller circular chromosomes. Chromosomal recombination plays a vital role in genetic diversity. In prokaryotes and viruses.

+21. 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. The individual would have Down Syndrome and his/her karyotype would be written 47.XY or 47. XX (female) or 46 XY (male). the individual carrying the mutation is said to be aneuploid. rather than 2. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.XX. Euploid human karyotypes are 46. a extra copy of human chromosome 21).g.+21. Such individuals are called euploid and have the wild-type chromosome complement for the species. If the mutation involves only one or a few chromosomes in the genome (e. in which an individual has 3. reflecting their bacterial origins. An example of aneuploidy is trisomy 21.are also found in mitochondria and chloroplasts. 1. 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). .3 MUTATIONS IN CHROMOSOME NUMBER Normally. copies of chromosome 21.

longer-lasting attachment in this region. chromosomes are structurally highly condensed. along with special proteins. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. The microtubules then pull the chromatids apart toward the centrosomes. small) and the longer arms are called q arms (q follows p in the Latin alphabet. This compact form makes the individual chromosomes visible. During mitosis.Fig 1. In spite of their appearance. one of which is present on each sister chromatid. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. . which enables these giant DNA structures to be contained within a cell nucleus. a pair of sister chromatids attached to each other at the centromere. and they form the classic four arm structure.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). 1. the chromatin strands become more and more condensed. Once the cells have divided. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. so that each daughter cell inherits one set of chromatids. The shorter arms are called p arms (from the French petit. 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. q-g "grande"). A special DNA base sequence in the region of the kinetochores provides.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II.

6 kg (5. in two separate nuclei. bone marrow constitutes 4% of the total body mass of humans. which use the bone marrow vasculature as a conduit to the body's systemic circulation. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. In humans.7 lbs).1. On average. in adults weighing 65 kg (143 lbs). bone marrow in large bones produces new blood cells.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. organelles and cell membrane into two cells containing roughly equal shares of these cellular components.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. which divides the nuclei. bone marrow accounts for approximately 2. cytoplasm. It is generally followed immediately by cytokinesis. [1] Bone marrow is also a key component of the lymphatic system. . producing the lymphocytes that support the body's immune system CHAPTER 2 2. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day.

The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. forming single cells with multiple nuclei. For example. Even in animals.[1] Prokaryotic cells. The cell then divides in cytokinesis. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. . prophase. These stages are interphase. This occurs most notably among the fungi and slime moulds. which lack a nucleus. Because cytokinesis usually occurs in conjunction with mitosis. to produce two identical daughter cells which are still diploid cells. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. The process of mitosis is fast and highly complex. This accounts for approximately 10% of the cell cycle. anaphase and telophase. prometaphase.genetically identical to each other and to their parent cell. "mitosis" is often used interchangeably with "mitotic phase". for instance during certain stages of fruit fly embryonic development. Mitosis occurs only in eukaryotic cells and the process varies in different species. metaphase. where the nuclear envelope breaks down before the chromosomes separate. animals undergo an "open" mitosis. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. where chromosomes divide within an intact cell nucleus. but is found in various different groups. there are many cells where mitosis and cytokinesis occur separately. cytokinesis and mitosis may occur independently. divide by a process called binary fission. However.

The sister chromatids are held together by a specialized region of the chromosome known as the centromere. . 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. and together the two are called sister chromatids. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. This occurs during the S phase of interphase. Each chromosome now has an identical copy of itself.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. the parent cell must make a copy of each chromosome before mitosis. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function.

A new nuclear envelope forms around the separated sister chromosomes. Prokaryotic cells undergo a process similar to mitosis called binary fission. the nuclear envelope which segregates the DNA from the cytoplasm disassembles.the cell begins cytokinesis. As the cell elongates. In plant cells. so they are renamed to sister chromosomes. giving rise to two daughter cells. pulling apart the sister chromatids of each chromosome.In most eukaryotes. the daughter cells will construct a new dividing cell wall between each other. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. each sister chromatid is now considered a chromosome. However. In animal cells. 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. the parent cell will be split in half. separating the two developing nuclei. each with a replica of the original genome. Eventually. As a matter of convention. 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. corresponding sister chromosomes are pulled toward opposite ends. .

continues to grow as it duplicates its chromosomes (S). and finally it divides (M) before restarting the cycle. S (synthesis). All these phases in the interphase are highly regulated. During all three phases. and G2 (second gap). chromosomes are replicated only during the S phase. grows more and prepares for mitosis (G 2). It alternates with the much longer interphase.1 Preprophase In plant cells only.2. where the cell prepares itself for cell division. This is achieved through the formation of a phragmosome. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell .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. Thus. prophase is preceded by a pre-prophase stage. the cell grows by producing proteins and cytoplasmic organelles. However. the nucleus has to migrate into the center of the cell before mitosis can begin. mainly via proteins. 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.2. a cell grows (G1). In highly vacuolated plant cells. Interphase is divided into three phases: G1 (first gap). 2.

instead. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. 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. The cells of higher plants (such as the flowering plants) lack centrioles. after the nuclear membrane breaks down. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. The chromosomes have chromatin has condensed. and microtubules have invaded the nuclear Prophase space. In addition to phragmosome formation. Telophase: The decondensing chromosomes are surrounded by nuclear membranes. . This band marks the position where the cell will eventually divide. aligned at the metaphase plate. degraded. the pinched area is known as the cleavage furrow.division. Cytokinesis has already begun. Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. These microtubules can attach to kinetochores or they can interact with opposing microtubules.

The centrosome is the coordinating center for the cell's microtubules. At the onset of prophase. A cell inherits a single centrosome at cell division. chromatin condenses together into a highly ordered structure called a chromosome. the replicated chromosomes have two sister chromatids. Chromosomes are typically visible at high magnification through a light microscope. the genetic material in the nucleus is in a loosely bundled coil called chromatin. which are made of a pair of centrioles found in most eukaryotic animal cells. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell. which is replicated by the cell with the help of the nucleus before a new mitosis begins. bound together at the centromere by the cohesin protein complex. Although centrioles help organize microtubule assembly.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. Close to the nucleus are structures called centrosomes. they are not essential for the . The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. Since the genetic material has already been duplicated earlier in S phase. giving a pair of centrosomes.

formation of the spindle. undergo a variation called closed mitosis where the spindle forms inside the nucleus. or its microtubules are able to penetrate an intact nuclear envelope. Although the kinetochore structure and function are not fully understood. 2. such as algae or trichomonads. since they are absent from plants. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. on an average 20 ). Prometaphase is sometimes considered part of prophase. provides the pulling force necessary to later separate the chromosome's two chromatids. the motor activates. it is known that it contains some form of molecular motor.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. . A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook. coupled with polymerisation and depolymerisation of microtubules. This motor activity. When a microtubule connects with the kinetochore. Fungi and some protists.2. This is called open mitosis. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle. one attached at each chromatid. and it occurs in most multicellular organisms. and centrosomes are not always used in mitosis. using energy from ATP to "crawl" up the tube toward the originating centrosome. When the spindle grows to sufficient length. kinetochore microtubules begin searching for kinetochores to attach to. Each chromosome forms two kinetochores at the centromere.

Metaphase comes from the Greek meaning "after. the kinetochore would be the "hook" that catches a sister chromatid or "fish". As a result. convene along the metaphase plate or equatorial plane. The centromeres of the chromosomes. . In certain types of cells. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". analogous to a tug-of-war between people of equal strength. the chromosomes come under longitudinal tension from the two ends of the cell. an imaginary line that is equidistant from the two centrosome poles. All chromosomes (blue) but one have arrived at the metaphase plate. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.3 Metaphase A cell in late metaphase. 2. in some sense." Microtubules find and attach to kinetochores in prometaphase.In the fishing pole analogy. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. only roughly lining up along the midline. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.

” or “re-”). At the end of anaphase. Two events then occur: first. 2. . The force that causes the centrosomes to move towards the ends of the cell is still unknown. the nonkinetochore microtubules elongate.” “back.” “against. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. which have now become distinct sister chromosomes. the cell proceeds to anaphase (from the Greek meaning “up. The signal creates the mitotic spindle checkpoint. These sister chromatids. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). These two stages are sometimes called early and late anaphase. the proteins that bind sister chromatids together are cleaved. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. 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. Early anaphase is usually defined as the separation of the sister chromatids. Next. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. allowing them to separate.

using fragments of the parent cell's nuclear membrane. pinching off the separated nuclei. with the cleavage furrow being clearly visible Cytokinesis is often mistakenly thought to be the final part of telophase. but rather a separate process. Corresponding sister chromosomes attach at opposite ends of the cell.2. A new nuclear envelope. unfold back into chromatin.5 Cytokinesis Cilliate undergoing cytokinesis. 2. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. Cytokinesis is technically not even a phase of mitosis. now surrounded by new nuclei. but cell division is not yet complete. At telophase. Both sets of chromosomes. the nonkinetochore microtubules continue to lengthen. Mitosis is complete. It "cleans up" the after effects of mitosis. cell . elongating the cell even more. forms around each set of separated sister chromosomes. cytokinesis is a separate process that begins at the same time as telophase.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. necessary for completing cell division. In animal cells. however. In both animal and plant cells.

each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. This is the basis of the development of a multicellular body from a single cell i.3 Cell replacement In some parts of body. 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.2 Development and growth The number of cells within an organism increases by mitosis. 2.1Significance Mitosis is important for the maintenance of the chromosomal set. Similarly. 2. zygote and also the basis of the growth of a multicellular body.5.e. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. 2. separating the two nuclei. cells are constantly sloughed off and replaced by new ones.g.5. e. which move along microtubules to the middle of the cell. The end of cytokinesis marks the end of the M-phase..5.4 Regeneration . The phragmoplast is a microtubule structure typical for higher plants. skin and digestive tract.division is also driven by vesicles derived from the Golgi apparatus. Each daughter cell has a complete copy of the genome of its parent cell. whereas some green algae use a phycoplast microtubule array during cytokinesis.5. New cells are formed by mitosis and so are exact copies of the cells being replaced.

5. a condition often associated with cancer. and the latter cell having only one chromosome (the homologous chromosome). sea star regenerates its lost arm through mitosis. a condition known as trisomy. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). These cells are considered aneuploid. they fail to complete cell division and retain both nuclei in one cell. For example. Occasionally when cells experience nondisjunction. the process may go wrong.Some organisms can regenerate their parts of bodies. The same division happens during asexual reproduction or vegetative propagation in plants. The production of new cells is achieved by mitosis.5. 2. Mitosis continues in the cells of bud and it grows into a new individual. the hydra reproduces asexually by budding. . In non-disjunction. especially during early cellular divisions in the zygote. resulting in binucleated cells.7 Consequences of errors Although errors in mitosis are rare.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. The cells at the surface of hydra undergo mitosis and form a mass called bud. a chromosome may fail to separate during anaphase. 2. For example. a condition known as monosomy. One daughter cell will receive both sister chromosomes and the other will receive none.

As long as these tumours remain in their original location they are called benign tumours. All cells have genes that control the timing and number of mitosis. It results in the synthesis of execessive tissue growths. sometimes mutuations occur in such genes and cells continue to divide. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. causing deletion. Occasionally. chromosomes may become damaged. but in reverse orientation. causing translocation. causing inversion. . and chromosomes are jostled constantly by probing microtubules. It results in abnormal cell growth. Benign tumours are not harmful as soon as they are not moving. it results in the formation of Tumors. It may reattach to the original chromosome.Mitosis is a demanding process for the cell. This phenomenon is called metastasis or spreading of disease. The fragment may incorrectly reattach to another. Now what happens is that cell abnormally continue to divide at a single place. its organelles disintegrate and reform in a matter of hours. When tissues more than the requirement are synthesized in a single organ. which goes through dramatic changes in ultrastructure. The effect of these genetic abnormalities depends on the specific nature of the error. As soon as they start to move and invade other cells there are said to be malignant tumours. non-homologous chromosome. Such tumours can send cancer cells to other parts in body where new tumours may form. Errors in the control of mitosis may cause cancer. An arm of the chromosome may be broken and the fragment lost. Or. it may be treated erroneously as a separate chromosome. causing chromosomal duplication.

is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. Early events of metaphase can .6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. align in the middle of the cell before being separated into each of the two daughter cells. An example of a cell that goes through endomitosis is the megakaryocyte. only roughly lining up along the middleline. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. 2. Preceded by events in prometaphase and followed by anaphase. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.7 Metaphase Metaphase. analogous to a tug of war between equally strong people. an imaginary line that is equidistant from the two centrosome poles. from the ancient Greek(between) and (stage). chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. resulting in cells with many copies of the same chromosome occupying a single nucleus. This process may also be referred to as endoreduplication and the cells as endoploid. In certain types of cells.2. Metaphase accounts for approximately 4% of the cell cycle's duration. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). carrying genetic information.

often with Giemsa (G banding) or Quinacrine. Normal metaphase spreads are used in . This would be accomplished by regulation of the anaphase-promoting complex. Metaphase chromosomes make the classical picture of chromosomes (karyotype).coincide with the later events of prometaphase. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. Chromosomes are condensed(Thickened) and highly coiled in metaphase. securin. 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. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. 2. which makes them most suitable for visual analysis. and separase. when every kinetochore is properly attached to a bundle of microtubules. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Staining of the slides. produces a pattern of in total up to several hundred bands. Such a signal creates the mitotic spindle checkpoint. 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). Only after all chromosomes have become aligned at the metaphase plate. For classical cytogenetic analyses. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. does the cell enter anaphase.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies.

losses of chromosomal segments or translocations. . 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. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. for example. such as bcr-abl in chronic myelogenous leukemia.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments.

and any other physical characteristics. and the results may be used in evolutionary biology and medicine. taxonomic relationships. Karyogram of human male using Giemsa staining. Karyotypes can be used for many purposes. such as. There may. The study of karyotypes is important for cell biology and genetics. Attention is paid to their length. ordered by size and position of centromere for chromosomes of the same size. in normal diploid organisms. in humans 2n = 46. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. The study of whole sets of chromosomes is sometimes known as karyology. Karyotypes describe the number of chromosomes. any differences between the sex chromosomes. banding pattern. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). cellular function. autosomal chromosomes are present in two copies. or may not. The term is also used for the complete set of chromosomes in a species. or an individual organism.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. the position of the centromeres. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. . Thus. and what they look like under a light microscope. So. [4] The preparation and study of karyotypes is part of cytogenetics. be sex chromosomes. to study chromosomal aberrations. and to gather information about past evolutionary events. 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 discoverer of mitosis. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. in contrast to their genic contents. and he correctly insisted on humans having an XX/XY system. in 1882. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. The next stage took place after the development of genetics in the early 20th century. Using cells in culture 2. these results were quite remarkable. The name was coined by another German anatomist. Considering their techniques. which swells them and spreads the chromosomes .1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. von Waldeyer in 1888. He revised his opinion later from 46 to 48. Pretreating cells in a hypotonic solution.3. concluding an XX/XO sex determination mechanism. 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. The subsequent history of the concept can be followed in the works of Darlington and White. at first favoring 46. Their behavior in animal (salamander) cells was described by Walther Flemming. New techniques were needed to definitively solve the problem: 1. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes.

is applied after cells have been arrested during cell division by a solution of colchicine. Usually. the great apes have 48 chromosomes. such as Giemsa. Human chromosome 2 was formed by a merger of ancestral chromosomes. reducing the number. [16] Sometimes observations may be made on non-dividing (interphase) cells.3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. 3. a suitable dye. Arresting mitosis in metaphase by a solution of colchicines 4. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Rather interestingly. For humans.2. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. The sex of an unborn fetus can be determined by observation of interphase cells. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.2 Observations on karyotypes 3. 3.2 Observations Six different characteristics of karyotypes are usually observed and compared: .1 Staining The study of karyotypes is made possible by staining.2.

and mainly consists of genetically inactive repetitive DNA sequences. Differences in degree and distribution of heterochromatic regions. 5. type. indicating tighter packing. Differences in the position of centromeres. 2. which (when they occur) are small bodies attached to a chromosome by a thin thread. . 3. permitting its loss without penalty to the organism (the dislocation hypothesis). A full account of a karyotype may therefore include the number. This feature probably reflects different amounts of DNA duplication. but the genes have been mostly translocated (added) to other chromosomes. Heterochromatin stains darker than euchromatin. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes). faba chromosomes are many times larger.1. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Differences in absolute sizes of chromosomes. 6. shape and banding of the chromosomes. Differences in number and position of satellites. This is brought about by translocations. as well as other cytogenetic information. 4. Humans have one pair fewer chromosomes than the great apes. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths. both have six pairs of chromosomes (n=6) yet V.

Any variation from the standard karyotype may lead to developmental abnormalities. which are highly variable.Variation is often found: 1. XY. Between members of a population (chromosome polymorphism) 4. males have both an X and a Y chromosome denoted 46.3 The human karyotype Most (but not all) species have a standard karyotype. the same cannot be said for their karyotypes. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Between the sexes 2. 3. XX. 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. and in . Mosaics or otherwise abnormal individuals. Geographical variation between races 5. There is variation between species in chromosome number. Normal karyotypes for females contain two X chromosomes and are denoted 46.

portions of the chromosomes are cast away in particular cells. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost.1 Changes during development Instead of the usual gene repression. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. In A. found in some copepods and roundworms such as Ascaris suum. Although much is known about karyotypes at the descriptive level. In a review. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. or other kinds of visible adjustment to the karyotype. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species.. In this process. some organisms go in for large-scale elimination of heterochromatin. despite their construction from the same macromolecules. Chromatin diminution (founding father: Theodor Boveri). In some species.. Godfrey and Masters conclude: "In our view. In some cases there is even significant variation within species. entire chromosomes are eliminated during development.detailed organization.3. despite many careful investigations. But. This variation provides the basis for a range of studies in evolutionary cytology.. used in conjunction with other phylogenetic data. the general significance of karyotype evolution is obscure. it is quite unclear what the general significance might be. Chromosome elimination. which were previously inexplicable. "We have a very poor understanding of the causes of karyotype evolution. 3.. as in many sciarid flies. .

In marsupials it is always the paternal X which is inactivated. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. In human females some 15% of somatic cells escape inactivation.suum. "They simply could not believe what they saw. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). 3. all the somatic cell precursors undergo chromatin diminution. the high record would be somewhere amongst the ferns.. Muntiacus reevesi.. they were astonished to find it had female = 6. The existence of supernumerary or B chromosomes .3. was found to be 46. Muntiacus muntjak. They kept quiet for two or three years because they thought something was wrong with their tissue culture.. the inactivation is random as between the two Xs. When they looked at the karyotype of the closely related Indian muntjac. which was investigated by Kurt Benirschke and his colleague Doris Wurster. Xinactivation. where the haploid n = 1. all telocentric. The low record is held by the nematode Parascaris univalens.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. male = 7 chromosomes. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. In placental mammals. The diploid number of the Chinese muntjac. 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.

Haplo-diploidy. due to the presence of five acrocentric chromosome pairs (13. where there are more than two sets of homologous chromosomes in the cells. Polyploidy in lower plants (ferns. and aneuploids are another example. 21 and 22).4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. where one sex is diploid. and the other haploid. about 70%. FN. but in grasses the average is much higher. It is a common arrangement in the Hymenoptera. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. 15.Endopolyploidy occurs when in adult differentiated . occurs mainly in plants. horsetails and psilotales) is also common.means that chromosome number can vary even within one interbreeding population. Humans have FN = 82. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. Polyploidy. though in this case they would not be regarded as normal members of the population.3.3 Fundamental number The fundamental number. FN ≤ 2n. Polyploidy in animals is much less common. 3. It has been of major significance in plant evolution according to Stebbins. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. and in some other groups. but it has been significant in some groups. Thus. 3. 14.

endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). Down syndrome and Turner syndrome are examples of this. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. 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. the daughter chromosomes separating from each other inside an intact nuclear membrane.tissues the cells have ceased to divide by mitosis. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. See palaeopolyploidy for the investigation of ancient karyotype duplications. The cells do not always contain exact multiples (powers of two).5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. In many instances. 3. it is diverse and complex. but the nuclei contain more than the original somatic number of chromosomes. Abnormalities in chromosome number usually cause a defect in development. 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. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. .

3. When this happens. and Crocus.500 sq mi (17. 5. the chromosome number is variable from one individual to another. [41] Closer to home.000 km2). 4.Aneuploidy may also occur within a group of closely related species. the European shrew Sorex araneus. Human chromosome 2 was formed by a merger of ancestral chromosomes. where every number from x = 3 to x = 15 is represented by at least one species. some mantids of the genus Ameles. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. living from rainforests to . Well-researched examples are the ladybird beetle Chilocorus stigma. where the gametic (= haploid) numbers form the series x = 3. 3. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast.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. Classic examples in plants are the genus Crepis. reducing the number. 6. that the two chromosome morphs are adapted to different habitats. In about 6.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. the great apes have 24x2 chromosomes whereas humans have 23x2. and 7.

show a clear "flow" of species from older to newer islands. at least into the Cretaceous. it is more likely to have been a group from the same species. Drosophila and Scaptomyza. The inversions. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. the best-studied group of Hawaiian drosophilids. the present islands date from 0. Although it would be possible for a single gravid female to colonise an island. make it possible to see which species are closely related. There are also cases of colonization back to older islands. gene arrangements are visible in the banding patterns of each chromosome. Using K-Ar dating. in the family Drosophilidae. especially inversions. All of the native Drosophila and Scaptomyza species in Hawaii have apparently descended from a single ancestral species that colonized the islands. but these are much less frequent. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. when plotted in tree form (and independent of all other information). and skipping of islands. . probably 20 million years ago. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. The results are clear. Chromosome rearrangements.4 million years ago (mya) (Mauna Kea) to 10mya (Necker).subalpine meadows. which can be dated to 30 mya. The polytene banding of the 'picture wing' group. Previous islands now beneath the sea (guyots) form the Emperor Seamount Chain. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. In a sense.

3.7. This method will normally produce 300-400 bands in a normal. • T-banding: visualize telomeres. R-banding is the reverse of G-banding (the R stands for "reverse"). • Q-banding is a fluorescent pattern obtained using quinacrine for staining.There are other animals and plants on the Hawaiian archipelago which have undergone similar. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). adaptive radiations. if less spectacular. The light regions tend to be euchromatic.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.7 Depiction of karyotypes 3. It yields a series of lightly and darkly stained bands . . • • C-banding: Giemsa binds to constitutive heterochromatin. human genome. The pattern of bands is very similar to that seen in G-banding.the dark regions tend to be heterochromatic. early-replicating and GC rich. so it stains centromeres. late-replicating and AT rich.

the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. Each chromosome has a characteristic banding pattern that helps to identify them. and the long arm on the bottom. is used to stain bands on the chromosomes. less frequently Quinacrine. Cri du chat syndrome involves a deletion on the short arm of . 3. In the "classic" (depicted) karyotype. In addition. respectively. both chromosomes in a pair will have the same banding pattern. a dye. For example. Giemsa is specific for the phosphate groups of DNA. Some karyotypes call the short and long arms p and q.7.• Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. Karyotypes are arranged with the short arm of the chromosome on top. This yields a dark region where the silver is deposited. Quinacrine binds to the adeninethymine-rich regions. denoting the activity of rRNA genes within the NOR. often Giemsa (G-banding).2 Classic karyotype cytogenetics Karyogram from a human female lymphocyte probed for the Alu sequence using FISH.

3. It is written as 46.3 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.5p-.chromosome 5. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. a combinatorial labeling method is used to generate many different colors. This method is also known as virtual karyotyping. The critical region for this syndrome is deletion of 15.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.del(5)(p15. 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. allowing the visualization of the individually colored chromosomes.7. Because there are a limited number of spectrally-distinct fluorophores. which is written as 46. . Image processing software then assigns a pseudo color to each spectrally different combination. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.XX.2) 3. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.2.XX.


Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18. the most common male chromosomal disease. Down syndrome. as in the presence of extra or missing chromosomes. trisomy 9 and trisomy 16. although they generally do not survive to birth. is caused by trisomy of chromosome 21. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. or structural. a common chromosomal disease. including . Also documented are trisomy 8. Some disorders arise from loss of just a piece of one chromosome. • • Patau syndrome is caused by trisomy of chromosome 13. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. in which three copies of a chromosome are present instead of the usual two. Numerical abnormalities. large-scale deletions or duplications. trisomies. X0). otherwise known as 47. are common numerical abnormalities. Structural abnormalities often arise from errors in homologous recombination. often occur as a result of nondisjunction during meiosis in the formation of a gamete.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. also known as aneuploidy. XXY is caused by an extra X chromosome. Klinefelter syndrome. as in derivative chromosome.4. X or 45. inversions. translocations.

• Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. • • Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. 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. caused by abnormal formation of the larynx. a deletion of the paternal genes. The name comes from the babies' distinctive cry. a deletion of the maternal genes. from a truncated short arm on chromosome 5. There are many types of chromosome anomalies. example of imprinting disorder. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. A chromosome anomaly may be detected or confirmed in this manner. example of imprinting disorder. 1p36 Deletion syndrome. .• Cri du chat (cry of the cat). numerical and structural anomalies. A chromosome anomaly. They can be organized into two basic groups. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes. one well-documented example is the Philadelphia chromosome. from the loss of part of the short arm of chromosome 1. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia.

which is caused by partial deletion of the short arm of chromosome 4. There are two main types of translocations. etc. also called the terminal 11q deletion disorder. 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 17.4. In humans an example of a condition caused by a numerical anomaly is Down Syndrome.). This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). 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 entire chromosome has . In a Robertsonian translocation. Tetrasomy. segments from two different chromosomes have been exchanged. In a reciprocal translocation. 4. an X. Known disorders in humans include Wolf-Hirschhorn syndrome. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. Duplications: A portion of the chromosome is duplicated. and Jacobsen syndrome.3 Structural abnormalities When the chromosome's structure is altered. • • Translocations: When a portion of one chromosome is transferred to another chromosome. resulting in extra genetic material. rather than two).

as well . This is why chromosome studies are often performed on parents when a child is found to have an anomaly. therefore the genetic material is inverted. turned upside down and reattached.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. 21 and 22. 14.attached to another at the Centromere . especially the chromosomes. and are therefore initially not inherited. can happen after conception. Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage. This can happen with or without loss of genetic material. other cytogenetic banding techniques. • Rings: A portion of a chromosome has broken off and formed a circle or ring. Chromosome anomalies can be inherited from a parent or be "de novo". resulting in Mosaicism (where some cells have the anomaly and some do not). Some anomalies. 4. 15. 4. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell.in humans these only occur with chromosomes 13. • Inversions: A portion of the chromosome has broken off. It includes routine analysis of G-Banded chromosomes. They often lead to an increased tendency to develop certain types of malignancies. the anomaly is present in every cell of the body. however. Therefore.

He revised his opinion later from 46 to 48. in contrast to their genic contents. the discoverer of mitosis. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). in 1882. at first favoring 46. New techniques were needed to definitively solve the problem: 1. Their behavior in animal (salamander) cells was described by Walther Flemming. von Waldeyer in 1888. Considering their techniques. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. 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. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. these results were quite remarkable.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Using cells in culture 2. which swells them and spreads the chromosomes . The next stage took place after the development of genetics in the early 20th century. Pre-treating cells in a hypotonic solution. The name was coined by another German anatomist. concluding an XX/XO sex determination mechanism. 4. and he correctly insisted on man having an XX/XY system.

6 Applications in biology 4. Arresting mitosis in metaphase by a solution of colchicine 4. 4. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.3. reducing the number.6. persimilis from wild populations in California and neighboring states.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. Rather interestingly. Using Painter's technique they studied the polytene .2 Natural populations of Drosophila In the 1930s. During her cytogenetic work.6. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. In 1931. the great apes have 48 chromosomes. 4. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Human chromosome 2 was formed by a merger of ancestral chromosomes. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. McClintock discovered transposons. a find which eventually led to her Nobel Prize in 1983. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D.

Evidence rapidly accumulated to show that natural selection was responsible. as they would if selectively neutral. as with most polymorphisms. Dobzhansky bred populations in population cages. cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy. In 1959. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. which enabled feeding. breeding and sampling whilst preventing escape. such as Down's syndrome. . This had the benefit of eliminating migration as a possible explanation of the results. Down syndrome is also referred to as trisomy 21.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. 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. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. 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. but adjust to certain frequencies at which they become stabilised.7 Human abnormalities and medical applications In the event of procedures which allowed easy enumeration of chromosomes. In some congenital disorders. It was found that the various chromosome types do not fluctuate at random. discoveries were quickly made related to aberrant chromosomes or chromosome number. 4. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. Using a method invented by L'Heretier and Teissier.

Pennsylvania. Many other sex chromosome combinations are compatible with live birth including XXX. Thirteen years later. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. an additional X chromosome in a male. is used today as a diagnostic for CML. 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. and XXXX. .Other numerical abnormalities discovered include sex chromosome abnormalities. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome. In 1960. with the development of more advanced techniques. which is required in normal females to compensate for having two copies of the chromosome. This abnormal chromosome was dubbed the Philadelphia chromosome .as both scientists were doing their research in Philadelphia. has Klinefelter's Syndrome. Peter Nowell and David Hungerford [17] discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). Identification of the Philadelphia chromosome by cytogenetics. in addition to other tests. Not all genes on the X Chromosome are inactivated. XYY. resulting in 47 total chromosomes.

Deletion syndromes such as DiGeorge syndrome. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps.FIG Advent of banding techniques In the late 1960s. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. 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. 4. 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.8 Beginnings of molecular cytogenetics . Caspersson developed banding techniques which differentially stain chromosomes. Deletions within one chromosome could also now be more specifically named and understood. and elongation techniques for all culture types that allow for higher resolution banding.

While radioisotope-labeled probes had been hybridized with DNA since 1969. movement was now made in using fluorescent labeled probes. CHAPTER 5 Techniques 5.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. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). cloned and studied in ever greater detail.1 Karyotyping . This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely.

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,

test and measurement. and FORTRAN. financial modeling and analysis. For congenital problems usually 20 metaphase cells are scored. data analysis. and numerical computation. and computational biology.generally between 200 and 1000 cells are counted and scored. You can use MATLAB in a wide range of applications. C++. Using MATLAB. CGH and Single nucleotide polymorphism-arrays. data visualization. CHAPTER 6 MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. including signal and image processing. control design. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. you can solve technical computing problems faster than with traditional programming languages. such as C. communications. . such as comparative genomic hybridization arrays. 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.

It enables fast development and execution. These effects must be compensated to improve the results of the pairing algorithm. In many cases. The image processing step is composed of the following operations. As a result. 6.MATLAB provides a number of features for documenting and sharing your work. 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. one line of MATLAB code can often replace several lines of C or C++ code.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. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. You can integrate your MATLAB code with other languages and applications. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. MATLAB eliminates the need for ‘for’ loops. With the MATLAB language. specifying data types. and distribute your MATLAB algorithms and applications. such as declaring variables. and allocating memory. .

Therefore. geometrical and dimensional differences must be removed. 2) Geometrical compensation—The geometric compensation. To compare chromosomes from a band pattern point of view. To compensate for this inhomogeneity. or at least attenuated. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast. 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).1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. 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. the spatially scaled images are histogram equalized.2 Concepts used in this phase 1) Image conversion 2) Denoising . 6. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis.

You can perform certain conversions just using MATLAB syntax.I). you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. MATLAB simply applies the filter to the indices in the indexed image matrix. if you want to filter a color image that is stored as an indexed image. RGB = cat (3. as is appropriate.3) Edge detection 4) Two dimensional convolutions. 6. green.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. In addition to these image type conversion functions. For example. .2. you must first convert it to true color format. listed in the following table.I. For example. so the image displays as shades of gray. When you apply the filter to the true color image. and the results might not be meaningful. For example. The resulting true color image has identical matrices for the red.I. If you attempt to filter the indexed image. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. there are other functions that return a different image type as part of the operation they perform. MATLAB filters the intensity values in the image. and blue planes.

6. we may expect errors to occur in the image signal.2. and hence the type of noise on the image. and we shall look at some of the more straightforward of them. .3 Two dimensional convolutions C = conv2(A.parameters. The general Matlab command for finding edges is edge(image. If an image is being sent electronically from one place to another. via satellite or wireless transmission. to recognize or classify objects. or through networked cable. Cleaning an image corrupted by noise is thus an important area of image restoration.'method'.2.5 Edge detection Edges contain some of the most useful information in an image.4 Denoising We may define noise to be any degradation in the image signal. caused by external disturbance. If one of these matrices describes a two-dimensional finite impulse response . We may use edges to measure the size of objects in an image.B) computes the two-dimensional convolution of matrices A and B. Usually we know what type of errors to expect. hence we can choose the most appropriate method for reducing the effects. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. There is a large number of edge finding algorithms in existence. . ) Where the parameters available depend on the method used 6. to isolate particular objects from their background. 6.

The indices of the center element of B are defined as floor(([mb C = conv2(hcol. The size of matrices.'sobel'). the other matrix is filtered in two dimensions. this case is the same as C = conv2(hcol*hrow. minus one.(FIR) filter.7). convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns. rgb2gray im2bw(im..nb].[3 3])..hrow. That is.bmp'). imedfilt2(im1.'shape') subsection of the two-dimensional convolution.na+nb-1].na] and the size of B is [mb. 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.A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. if the size of then the size of C is [ma+mb-1. nb]+1)/2). edge(im1.. C = conv2(.0. .A). If hcol is a column vector and hrow is a row vector.

double(msk)). n1(x1. [r.imy).j)==L_number(k) flag=1. Index=1.2). bwlabel(B.[imx. flag=0. MODULE 2 clc [m.y1)=255. nzeros(imx. L_number=zeros(mx. Msk conv2(double(BW). rc = [r c]. y1=rc(i.1).j)~=0 for k=1:mx if L(i. for i=1:m for j=1:n if L(i. .n]=size(L). mx=max(max(L)).8).imy]=size(BW). [sx sy]=size(rc).1). for i=1:sx x1=rc(i.c] = find(L==22).

50. n1=zeros(imx.32.14.35. Test_number=[3.j). end.8.4. end end L_number.6.c] = find(L==L_number((Test_number(x)))). n1(x1.22.2).41.48.y1)=255.40.62. Index=Index+ . 36.end end if flag~=1 L_number(Index)=L(i.56.27.31.imy). for x=1:46 [r.11. for i=1:sx x1=rc(i.]. end flag=0.51.imshow(n1.33. rc = [r c].20.[]).19.1).29.49. [sx sy]=size(rc). y1=rc(i.55. end %h=figure.39.65.9.

5*graythresh(skel)). [m n]=size(BW1). BW=im2bw(f). Arm_length=zeros(46. for i=1:46 f=imread(strcat(num2str(i). BW=double(BW).'.1). Circumference_sum=0.'spur'. BW1=edge(BW.'canny').Inf). Arm_length_sum=0. s1=bwmorph(s.end Circumference=zeros(46. skel=im2bw(skel.8).1.1).'skel'. for x=1:m for y=1:n if BW1(x. s=bwmorph(skel.bmp')).1). Area=zeros(46. skel=im2double(f).y)==1 Circumference_sum=Circumference_sum+1. f=imcomplement(f). . end end end Circumference(i)=Circumference_sum.

Arm_length.[m n]=size(s1). end end end Arm_length(i)=Arm_length_sum. Area_sum=0. for x=1:m for y=1:n if s1(x. [m n]=size(BW). . BW=im2bw(f). for x=1:m for y=1:n if BW(x.y)==1 Arm_length_sum=Arm_length_sum+1. end Circumference. end end end Area(i)=Area_sum.y)==1 Area_sum=Area_sum+1.

end end Pair. Pair(i.2)=i+1. Pair(i.2)=i. .2)==46 Pair(46. 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)). Pair=zeros(46. 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)). Pair(46.1)=i.1)=i.Area. Pair(i. end end end for i=1:45 if Pair(i. Pair(i.2).2)=j.1)=46.

1)==delete(j) flag=1.'.figure_flag). for i=1:46 for j=1:46 if Pair(i.2. figure_flag=1. delete(figure_flag)=Pair(i. .1).2). figure_flag=figure_flag+1. if figure_flag~=47 subplot(23. end flag=0. end end if flag~=1 if figure_flag~=47 subplot(23. imshow(f2).figure_flag).bmp')). end f1=imread(strcat(num2str(Pair(i.2. end f2=imread(strcat(num2str(Pair(i. imshow(f1).bmp')). flag=0.1)).'.2)).delete=zeros(46. figure_flag=figure_flag+1.

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. in the scope of karyotyping process used in cytogentic analysis. 2) feature extraction from the processed images . Copenhagen. 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.end CONCLUTION In this paper. and Philadelphia. dimensions and banding profiles. to improve the discriminative power of the pairing algorithm with respect to the the G-banding pattern. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. The proposed algorithm is based on the traditional features extracted from the karyogram. such as. plus a new one. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. based on the MI.

The features extracted from the processed images discriminate each pair with respect to their size. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts.working within an 8-D feature space. shape. achieves a 70. 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 . from the chromosomes in the training set. The training process consists in the estimation of each vector of coefficient . and finally. This normalization is needed to make it possible the band pattern comparison between chromosomes. shape and band pattern. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature. Here. 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. Tests using 19 karyograms based on bone marrow cells. the romosome images. 4) pairing. extracted from the unordered karyogram.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms.10% mean classification rate. 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). 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. In the image processing step. and band pattern. and to normalize their dimensions.characterizing the size. are processed in order to compensate for geometrical and intensity distortions. 3) training of a classifier (performed once) where similarity among chromosomes are characterized.

despite the low quality of this type of chromosomes. In addition. In fact. and from which it is possible to extract additional features. called LK1 . The results presented in this paper are promising.g. a new chromosome dataset with 9200 chromosomes from bone marrow cells. Copenhagen. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. a 76. or Philadelphia. Using 27 karyograms andworking with a limited number of classes (≤ 8). amean classification rate larger than 93% was obtained in all experiments.performance of the classifier.10% classification ratewas obtained. whose images are of significantly higher quality. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. This dataset was made publicly available [29]. REFERENCES . centromere position. such as Edinburgh.. e. presenting a uniform level of condensation. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. Executing the algorithm on a higher quality dataset.

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R.L. 1996.S. PNAS 97. In The ACT Cytogenetics Laboratory Manual 2nd ed. ^ Painter T.^ a b Gustashaw K. Kinetophore reproduction theory may explain rapid chromosome evolution. 1991.gov/articlerender. & Tobler H. 1923. ^ Godfrey L. 2001. Chromosome stains. NY. Hereditas 42.J. 1998. p85-6 18. ^ Goday C. ^ Tjio J. ^ Stebbins G.M. Bioessays23: 242–250. Oxford. 291-336. Barch. ^ A preparation which includes the dyes Methylene Blue. Chromosome elimination in sciarid flies. Bioessays18: 133–138. 17. London. The Association of Cytogenetic Technologists. 1971. ^ Maynard Smith J. 21. ^ Müller F.http://www. 15. and Masters J. M. Eosin Y and Azure-A. ed. 1-6. and Esteban M.12.R. Zoology 37.^ a b Hsu T.H & Levan A.nih. 2000. 2nd ed. Studies in mammalian spermatogenesis II.C 16. p218-9 20.B. The chromosome number of man. The spermatogenesis of humans. New York. Chromosomal evolution in higher plants.pubmedcentral. Raven Press. 9821– 9823. 1979. J.C. 13.C.fcgi?artid=34032 19. Chromatin diminution in nematodes. Human and mammalian cytogenetics: a historical perspective. Exp. 1956. Evolutionary genetics. Arnold. Springer-Verlag. 14. Bernard V. .

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