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

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

+21. reflecting their bacterial origins. Such individuals are called euploid and have the wild-type chromosome complement for the species. An example of aneuploidy is trisomy 21. XX (female) or 46 XY (male). If the mutation involves only one or a few chromosomes in the genome (e. 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. 1. copies of chromosome 21. 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). .g. 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.XX. The individual would have Down Syndrome and his/her karyotype would be written 47. the individual carrying the mutation is said to be aneuploid. a extra copy of human chromosome 21). Euploid human karyotypes are 46.are also found in mitochondria and chloroplasts.XY or 47. rather than 2.3 MUTATIONS IN CHROMOSOME NUMBER Normally.

q-g "grande"). 1. 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. chromosomes are structurally highly condensed. This is the only natural context in which individual chromosomes are visible with an optical microscope. This compact form makes the individual chromosomes visible. In spite of their appearance. . small) and the longer arms are called q arms (q follows p in the Latin alphabet.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. Once the cells have divided. During mitosis. one of which is present on each sister chromatid.Fig 1. which enables these giant DNA structures to be contained within a cell nucleus. so that each daughter cell inherits one set of chromatids. 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. the chromatids are uncoiled and DNA can again be transcribed. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. The shorter arms are called p arms (from the French petit. the chromatin strands become more and more condensed. along with special proteins. and they form the classic four arm structure. a pair of sister chromatids attached to each other at the centromere. longer-lasting attachment in this region.

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 constitutes 4% of the total body mass of humans. bone marrow accounts for approximately 2.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. which use the bone marrow vasculature as a conduit to the body's systemic circulation. In humans.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. . On average. which divides the nuclei.6 kg (5. bone marrow in large bones produces new blood cells.7 lbs). in adults weighing 65 kg (143 lbs). organelles and cell membrane into two cells containing roughly equal shares of these cellular components. cytoplasm. producing the lymphocytes that support the body's immune system CHAPTER 2 2. [1] Bone marrow is also a key component of the lymphatic system. in two separate nuclei.1. It is generally followed immediately by cytokinesis.

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

Each chromosome now has an identical copy of itself. . 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. Because each resultant daughter cell should be genetically identical to the parent cell. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. the parent cell must make a copy of each chromosome before mitosis. This occurs during the S phase of interphase. These two cells are identical and do not differ in any way from the original parent cell. The sister chromatids are held together by a specialized region of the chromosome known as the centromere.

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

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

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

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

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

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

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

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

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

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

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

chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. This process may also be referred to as endoreduplication and the cells as endoploid. An example of a cell that goes through endomitosis is the megakaryocyte.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.7 Metaphase Metaphase. from the ancient Greek(between) and (stage). 2. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores.2. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). Preceded by events in prometaphase and followed by anaphase. In certain types of cells. only roughly lining up along the middleline. Early events of metaphase can . analogous to a tug of war between equally strong people. resulting in cells with many copies of the same chromosome occupying a single nucleus. Metaphase accounts for approximately 4% of the cell cycle's duration. carrying genetic information. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. an imaginary line that is equidistant from the two centrosome poles.

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

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

Karyogram of human male using Giemsa staining. in normal diploid organisms. The term is also used for the complete set of chromosomes in a species. . cellular function. or an individual organism. and to gather information about past evolutionary events. The study of whole sets of chromosomes is sometimes known as karyology. and any other physical characteristics. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies. There may.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. banding pattern. Attention is paid to their length. autosomal chromosomes are present in two copies. or may not. the position of the centromeres. Karyotypes can be used for many purposes. in humans 2n = 46. be sex chromosomes. any differences between the sex chromosomes. taxonomic relationships. ordered by size and position of centromere for chromosomes of the same size. [4] The preparation and study of karyotypes is part of cytogenetics. The study of karyotypes is important for cell biology and genetics. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23). The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs. and the results may be used in evolutionary biology and medicine. Karyotypes describe the number of chromosomes. such as. Thus. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. to study chromosomal aberrations. So. and what they look like under a light microscope.

at first favoring 46. Using cells in culture 2. the discoverer of mitosis.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842.3. von Waldeyer in 1888. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. 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. in 1882. concluding an XX/XO sex determination mechanism. He revised his opinion later from 46 to 48. which swells them and spreads the chromosomes . Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. New techniques were needed to definitively solve the problem: 1. in contrast to their genic contents. and he correctly insisted on humans having an XX/XY system. Pretreating cells in a hypotonic solution. The subsequent history of the concept can be followed in the works of Darlington and White. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Their behavior in animal (salamander) cells was described by Walther Flemming. The next stage took place after the development of genetics in the early 20th century. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. Considering their techniques. The name was coined by another German anatomist. these results were quite remarkable.

Arresting mitosis in metaphase by a solution of colchicines 4.2. such as Giemsa. the great apes have 48 chromosomes.1 Staining The study of karyotypes is made possible by staining.2 Observations Six different characteristics of karyotypes are usually observed and compared: .2 Observations on karyotypes 3. The sex of an unborn fetus can be determined by observation of interphase cells. Human chromosome 2 was formed by a merger of ancestral chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. 3. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.3. For humans. is applied after cells have been arrested during cell division by a solution of colchicine. reducing the number. Usually. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes. a suitable dye. Rather interestingly.2. 3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. [16] Sometimes observations may be made on non-dividing (interphase) cells.

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

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

it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. found in some copepods and roundworms such as Ascaris suum. it is quite unclear what the general significance might be. In a review. In A. which were previously inexplicable. or other kinds of visible adjustment to the karyotype. despite many careful investigations. But.detailed organization. and it is clear that changes in karyotype organization have had effects on the evolutionary course of many species. In some cases there is even significant variation within species.. In some species. Chromatin diminution (founding father: Theodor Boveri). the general significance of karyotype evolution is obscure. In this process. portions of the chromosomes are cast away in particular cells. Godfrey and Masters conclude: "In our view. entire chromosomes are eliminated during development. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost.. despite their construction from the same macromolecules. . "We have a very poor understanding of the causes of karyotype evolution.1 Changes during development Instead of the usual gene repression.. some organisms go in for large-scale elimination of heterochromatin. 3.3. This variation provides the basis for a range of studies in evolutionary cytology. used in conjunction with other phylogenetic data. Chromosome elimination. Although much is known about karyotypes at the descriptive level. as in many sciarid flies.. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species.

The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). the inactivation is random as between the two Xs.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. Xinactivation. They kept quiet for two or three years because they thought something was wrong with their tissue culture. "They simply could not believe what they saw.. The diploid number of the Chinese muntjac. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes.3. In human females some 15% of somatic cells escape inactivation. In marsupials it is always the paternal X which is inactivated. they were astonished to find it had female = 6. where the haploid n = 1. 3. all telocentric.. which was investigated by Kurt Benirschke and his colleague Doris Wurster. The existence of supernumerary or B chromosomes . the high record would be somewhere amongst the ferns. male = 7 chromosomes.. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. Muntiacus reevesi. When they looked at the karyotype of the closely related Indian muntjac.. In placental mammals. The low record is held by the nematode Parascaris univalens. was found to be 46. Muntiacus muntjak. all the somatic cell precursors undergo chromatin diminution. thus the mammalian female is a mosaic in respect of her X chromosomes.suum. 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.

Thus. Polyploidy in animals is much less common. occurs mainly in plants.3 Fundamental number The fundamental number. Humans have FN = 82. where one sex is diploid. and in some other groups. horsetails and psilotales) is also common.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. 14. Polyploidy.3. 21 and 22). where there are more than two sets of homologous chromosomes in the cells. and aneuploids are another example. due to the presence of five acrocentric chromosome pairs (13.Endopolyploidy occurs when in adult differentiated . It is a common arrangement in the Hymenoptera. 3. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. and the other haploid. 3. though in this case they would not be regarded as normal members of the population.means that chromosome number can vary even within one interbreeding population. FN ≤ 2n. FN. about 70%. Haplo-diploidy. 15. but in grasses the average is much higher. It has been of major significance in plant evolution according to Stebbins. Polyploidy in lower plants (ferns. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. but it has been significant in some groups. of a karyotype is the number of visible major chromosomal arms per set 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. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted).5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. it is diverse and complex. and serves differentiation and morphogenesis in many ways.tissues the cells have ceased to divide by mitosis. In many instances. 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. This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost. 3. but the nuclei contain more than the original somatic number of chromosomes. Down syndrome and Turner syndrome are examples of this. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. Abnormalities in chromosome number usually cause a defect in development. See palaeopolyploidy for the investigation of ancient karyotype duplications. . the daughter chromosomes separating from each other inside an intact nuclear membrane.

and 7. the chromosome number is variable from one individual to another. the great apes have 24x2 chromosomes whereas humans have 23x2. Well-researched examples are the ladybird beetle Chilocorus stigma. reducing the number. 3. and Crocus. 6. When this happens. the European shrew Sorex araneus.500 sq mi (17. living from rainforests to . 4.6 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. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. [41] Closer to home. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. 3. where the gametic (= haploid) numbers form the series x = 3.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. 5. where every number from x = 3 to x = 15 is represented by at least one species.Aneuploidy may also occur within a group of closely related species. some mantids of the genus Ameles. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world.000 km2). In about 6. that the two chromosome morphs are adapted to different habitats. Classic examples in plants are the genus Crepis. Human chromosome 2 was formed by a merger of ancestral chromosomes.

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

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

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

allowing the visualization of the individually colored chromosomes. It is written as 46.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. Because there are a limited number of spectrally-distinct fluorophores. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Image processing software then assigns a pseudo color to each spectrally different combination. a combinatorial labeling method is used to generate many different colors. 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.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. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.5p-.del(5)(p15. This method is also known as virtual karyotyping.2) 3. which is written as 46. .XX. 3.XX.7. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.2.chromosome 5. The critical region for this syndrome is deletion of 15.


XXY is caused by an extra X chromosome. although they generally do not survive to birth. trisomy 9 and trisomy 16. trisomies. as in derivative chromosome. in which three copies of a chromosome are present instead of the usual two. the most common male chromosomal disease. are common numerical abnormalities. • • Edwards syndrome is caused by trisomy (three copies) of chromosome 18.4. Chromosomal abnormalities that lead to disease in humans include • • Turner syndrome results from a single X chromosome (45. X0). Numerical abnormalities. a common chromosomal disease.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. inversions. otherwise known as 47. Structural abnormalities often arise from errors in homologous recombination. Some disorders arise from loss of just a piece of one chromosome. as in the presence of extra or missing chromosomes. translocations. Also documented are trisomy 8. or structural. often occur as a result of nondisjunction during meiosis in the formation of a gamete. also known as aneuploidy. Klinefelter syndrome. • • Patau syndrome is caused by trisomy of chromosome 13. including . X or 45. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. Down syndrome. large-scale deletions or duplications. is caused by trisomy of chromosome 21.

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

4. etc. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. an X.3 Structural abnormalities When the chromosome's structure is altered. In a reciprocal translocation. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. rather than two). resulting in extra genetic material. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. Tetrasomy. which is caused by partial deletion of the short arm of chromosome 4. and Jacobsen syndrome. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. segments from two different chromosomes have been exchanged.). There are two main types of translocations. an entire chromosome has . 4. • • Translocations: When a portion of one chromosome is transferred to another chromosome. Known disorders in humans include Wolf-Hirschhorn syndrome. 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. Duplications: A portion of the chromosome is duplicated. In a Robertsonian translocation.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). also called the terminal 11q deletion disorder.

can happen after conception. turned upside down and reattached. They often lead to an increased tendency to develop certain types of malignancies. • Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. resulting in Mosaicism (where some cells have the anomaly and some do not). and are therefore initially not inherited. as well . Chromosome instability syndromes are a group of disorders characterized by chromosomal instability and breakage.in humans these only occur with chromosomes 13. Some anomalies.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. however. 4. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. 14. the anomaly is present in every cell of the body.attached to another at the Centromere . other cytogenetic banding techniques. • Rings: A portion of a chromosome has broken off and formed a circle or ring. Therefore. This can happen with or without loss of genetic material. 21 and 22.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. • Inversions: A portion of the chromosome has broken off. It includes routine analysis of G-Banded chromosomes. Chromosome anomalies can be inherited from a parent or be "de novo". therefore the genetic material is inverted. 15. 4. especially the chromosomes.

von Waldeyer in 1888.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. The next stage took place after the development of genetics in the early 20th century. and he correctly insisted on man having an XX/XY system. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. the discoverer of mitosis. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. concluding an XX/XO sex determination mechanism. The name was coined by another German anatomist. 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. these results were quite remarkable.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Their behavior in animal (salamander) cells was described by Walther Flemming. at first favoring 46. He revised his opinion later from 46 to 48. New techniques were needed to definitively solve the problem: 1. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. in 1882. in contrast to their genic contents. 4. Using cells in culture 2. Considering their techniques. Pre-treating cells in a hypotonic solution. which swells them and spreads the chromosomes . Painter in 1922 was not certain whether the diploid number of man was 46 or 48.

6 Applications in biology 4. 4. reducing the number.3.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist. persimilis from wild populations in California and neighboring states. Using Painter's technique they studied the polytene . It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes. In 1931. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. a find which eventually led to her Nobel Prize in 1983. Cutting up a photomicrograph and arranging the result into an indisputable karyogram.2 Natural populations of Drosophila In the 1930s. Arresting mitosis in metaphase by a solution of colchicine 4.6.6. McClintock discovered transposons. the great apes have 48 chromosomes. During her cytogenetic work. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. Rather interestingly.

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

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

8 Beginnings of molecular cytogenetics . Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps.FIG Advent of banding techniques In the late 1960s. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. Caspersson developed banding techniques which differentially stain chromosomes. Deletions within one chromosome could also now be more specifically named and understood. 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. and elongation techniques for all culture types that allow for higher resolution banding. 4. Deletion syndromes such as DiGeorge syndrome. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.

CHAPTER 5 Techniques 5. cloned and studied in ever greater detail. movement was now made in using fluorescent labeled probes.1 Karyotyping .In the 1980s. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated. advances were made in molecular cytogenetics. While radioisotope-labeled probes had been hybridized with DNA since 1969. 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,

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

It enables fast development and execution. The image processing step is composed of the following operations. . The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. 6. and allocating memory. As a result. 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. and distribute your MATLAB algorithms and applications. These effects must be compensated to improve the results of the pairing algorithm.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. 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. specifying data types. such as declaring variables.MATLAB provides a number of features for documenting and sharing your work. In many cases. MATLAB eliminates the need for ‘for’ loops. With the MATLAB language.

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

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

3 Two dimensional convolutions C = conv2(A. There is a large number of edge finding algorithms in existence. 6.5 Edge detection Edges contain some of the most useful information in an image. Usually we know what type of errors to expect. . These errors will appear on the image output in different ways depending on the type of disturbance in the signal. hence we can choose the most appropriate method for reducing the effects.'method'. The general Matlab command for finding edges is edge(image.6. via satellite or wireless transmission.4 Denoising We may define noise to be any degradation in the image signal. to isolate particular objects from their background. If one of these matrices describes a two-dimensional finite impulse response . and hence the type of noise on the image.2. . caused by external disturbance. We may use edges to measure the size of objects in an image. and we shall look at some of the more straightforward of them. If an image is being sent electronically from one place to another. to recognize or classify objects.B) computes the two-dimensional convolution of matrices A and B.2. Cleaning an image corrupted by noise is thus an important area of image restoration.parameters. we may expect errors to occur in the image signal. or through networked cable. ) Where the parameters available depend on the method used 6.

na] and the size of B is [mb.(FIR) filter. The indices of the center element of B are defined as floor(([mb C = conv2(hcol.na+nb-1]. rgb2gray im2bw(im. the other matrix is filtered in two dimensions..nb].hrow. C = conv2(.'shape') subsection of the two-dimensional convolution. this case is the same as C = conv2(hcol*hrow. convolves A first with the vector hcol along the rows and then returns a with the vector hrow along the columns.bmp'). imedfilt2(im1. edge(im1..A). If hcol is a column vector and hrow is a row vector. . minus one. The size of matrices.[3 3]).'sobel'). 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. if the size of then the size of C is [ma+mb-1.0. nb]+1)/2)..A) C in each dimension is equal to the sum of the corresponding dimensions of the input A is [ma. That is.7).

n1(x1.double(msk)).imy). [sx sy]=size(rc). Msk conv2(double(BW). nzeros(imx.n]=size(L). for i=1:m for j=1:n if L(i.8).1). for i=1:sx x1=rc(i.2). rc = [r c]. L_number=zeros(mx.y1)=255. mx=max(max(L)).1). MODULE 2 clc [m. y1=rc(i.[imx. [r.j)~=0 for k=1:mx if L(i. flag=0.c] = find(L==22). Index=1.imy]=size(BW). .j)==L_number(k) flag=1. bwlabel(B. end if flag~=1 L_number(Index)=L(i.60.29.imshow(n1.52.39. end %h=figure.7.48.[]). Index=Index+1. .y1)=255. for i=1:sx x1=rc(i.57. Test_number=[3.30. n1=zeros(imx.33.50.14.c] = find(L==L_number((Test_number(x)))). n1(x1.4.49. for x=1:46 [r. rc = [r c]. end flag=0.45.2).10.66].55. [sx sy]=size(rc). end end L_number.31.38. y1=rc(i.1).28.imy). end. 36.8.

1).'. BW1=edge(BW.y)==1 Circumference_sum=Circumference_sum+1.end Circumference=zeros(46.bmp')). for i=1:46 f=imread(strcat(num2str(i).1. end end end Circumference(i)=Circumference_sum. f=imcomplement(f). for x=1:m for y=1:n if BW1(x. BW=im2bw(f). Arm_length=zeros(46.1). s=bwmorph(skel.'spur'.'canny'). BW=double(BW). Circumference_sum=0. . s1=bwmorph(s. skel=im2bw(skel.8).1). [m n]=size(BW1).Inf). skel=im2double(f).5*graythresh(skel)). Area=zeros(46. Arm_length_sum=0.'skel'.

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

Pair(i. . 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)).2)=j.2).2)==46 Pair(46. Pair=zeros(46. Pair(i.1)=i.Area. 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)).2)=i+1.1)=i. Pair(i. end end Pair. Pair(46.2)=i.1)=46. end end end for i=1:45 if Pair(i.

2. if figure_flag~=47 subplot(23. end end if flag~=1 if figure_flag~=47 subplot(23.delete=zeros(46.2)). imshow(f1).1).'. . end f2=imread(strcat(num2str(Pair(i.2).figure_flag).2.'. figure_flag=figure_flag+1. delete(figure_flag)=Pair(i. end flag=0. end f1=imread(strcat(num2str(Pair(i. flag=0. imshow(f2).1)).bmp')). figure_flag=1. figure_flag=figure_flag+1.1)==delete(j) flag=1. for i=1:46 for j=1:46 if Pair(i.figure_flag).bmp')).

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

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

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

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