ABSTRACT

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.

CHAPTER 1

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

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

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

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

7 lbs). In humans. On average. which use the bone marrow vasculature as a conduit to the body's systemic circulation.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets. bone marrow in large bones produces new blood cells. which divides the nuclei. . The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day. cytoplasm. organelles and cell membrane into two cells containing roughly equal shares of these cellular components. in adults weighing 65 kg (143 lbs). Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones.1.6 kg (5. producing the lymphocytes that support the body's immune system CHAPTER 2 2. It is generally followed immediately by cytokinesis. bone marrow accounts for approximately 2. bone marrow constitutes 4% of the total body mass of humans. in two separate nuclei. [1] Bone marrow is also a key component of the lymphatic system.

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

Because each resultant daughter cell should be genetically identical to the parent cell. . the parent cell must make a copy of each chromosome before mitosis. 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. 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. This occurs during the S phase of interphase. and together the two are called sister chromatids. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs.

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

continues to grow as it duplicates its chromosomes (S). 2. grows more and prepares for mitosis (G 2). Thus. In highly vacuolated plant cells. However. the nucleus has to migrate into the center of the cell before mitosis can begin.2. mainly via proteins. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell . prophase is preceded by a pre-prophase stage.2 Phases of cell cycle and mitosis Interphase Fig 4 The cell cycle The mitotic phase is a relatively short period of the cell cycle.2. All these phases in the interphase are highly regulated. and finally it divides (M) before restarting the cycle. Interphase is divided into three phases: G1 (first gap). It alternates with the much longer interphase.1 Preprophase In plant cells only. the cell grows by producing proteins and cytoplasmic organelles. This is achieved through the formation of a phragmosome. S (synthesis). where the cell prepares itself for cell division. During all three phases. chromosomes are replicated only during the S phase. a cell grows (G1). and G2 (second gap). 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.

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

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

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

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

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

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

separating the two nuclei.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. whereas some green algae use a phycoplast microtubule array during cytokinesis.5. e. New cells are formed by mitosis and so are exact copies of the cells being replaced.e..5. Each daughter cell has a complete copy of the genome of its parent cell.5. 2. skin and digestive tract. Similarly. The phragmoplast is a microtubule structure typical for higher plants. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. cells are constantly sloughed off and replaced by new ones.3 Cell replacement In some parts of body.4 Regeneration .g. The end of cytokinesis marks the end of the M-phase. This is the basis of the development of a multicellular body from a single cell i. 2. which move along microtubules to the middle of the cell. zygote and also the basis of the growth of a multicellular body.division is also driven by vesicles derived from the Golgi apparatus. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. Following are the occasions in the lives of organism where mitosis happens: 2.5.

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

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

align in the middle of the cell before being separated into each of the two daughter cells. 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. Metaphase accounts for approximately 4% of the cell cycle's duration. Preceded by events in prometaphase and followed by anaphase. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. carrying genetic information. an imaginary line that is equidistant from the two centrosome poles.2. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. from the ancient Greek(between) and (stage). 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.7 Metaphase Metaphase. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). In certain types of cells. 2. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. analogous to a tug of war between equally strong people. Early events of metaphase can . only roughly lining up along the middleline. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase.

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

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. losses of chromosomal segments or translocations.methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. such as bcr-abl in chronic myelogenous leukemia. for example.

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

The name was coined by another German anatomist. the discoverer of mitosis. 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. at first favoring 46. 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. Pretreating cells in a hypotonic solution. Their behavior in animal (salamander) cells was described by Walther Flemming. and he correctly insisted on humans having an XX/XY system. He revised his opinion later from 46 to 48.3. concluding an XX/XO sex determination mechanism. these results were quite remarkable. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. Considering their techniques. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. The subsequent history of the concept can be followed in the works of Darlington and White. in contrast to their genic contents. in 1882.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. 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. von Waldeyer in 1888. which swells them and spreads the chromosomes . Using cells in culture 2.

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

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

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

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

3. Xinactivation. The existence of supernumerary or B chromosomes . Muntiacus reevesi. 3. 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. In placental mammals. The diploid number of the Chinese muntjac. where the haploid n = 1. was found to be 46. the high record would be somewhere amongst the ferns. Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. they were astonished to find it had female = 6.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.. all telocentric. Muntiacus muntjak.. which was investigated by Kurt Benirschke and his colleague Doris Wurster. When they looked at the karyotype of the closely related Indian muntjac. 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. The low record is held by the nematode Parascaris univalens. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes. male = 7 chromosomes.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac. In marsupials it is always the paternal X which is inactivated. thus the mammalian female is a mosaic in respect of her X chromosomes... In human females some 15% of somatic cells escape inactivation. all the somatic cell precursors undergo chromatin diminution.

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

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

reducing the number. Classic examples in plants are the genus Crepis.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. When this happens. 4.500 sq mi (17. the great apes have 24x2 chromosomes whereas humans have 23x2. 5. 3. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. [41] Closer to home. In about 6.000 km2). and Crocus. and 7. the chromosome number is variable from one individual to another. that the two chromosome morphs are adapted to different habitats. some mantids of the genus Ameles. 6. where every number from x = 3 to x = 15 is represented by at least one species. 3. where the gametic (= haploid) numbers form the series x = 3. Human chromosome 2 was formed by a merger of ancestral chromosomes.Aneuploidy may also occur within a group of closely related species. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups.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. There is some evidence from the case of the mollusc Thais lapillus (the dog whelk) on the Brittany coast. the European shrew Sorex araneus. Well-researched examples are the ladybird beetle Chilocorus stigma. living from rainforests to .

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

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

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

3.XX. which is written as 46. Image processing software then assigns a pseudo color to each spectrally different combination. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.XX.7.3 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. . a combinatorial labeling method is used to generate many different colors. allowing the visualization of the individually colored chromosomes.chromosome 5. Because there are a limited number of spectrally-distinct fluorophores. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Short sequences of DNA from specific loci all over the genome are isolated and enumerated.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.2) 3.5p-.del(5)(p15. The critical region for this syndrome is deletion of 15. It is written as 46. 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.2. This method is also known as virtual karyotyping.

CHAPTER 4 .

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

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

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

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

Using cells in culture 2. the discoverer of mitosis. 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. Painter in 1922 was not certain whether the diploid number of man was 46 or 48. these results were quite remarkable. which swells them and spreads the chromosomes . Considering their techniques. 4.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. and he correctly insisted on man having an XX/XY system. at first favoring 46.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Their behavior in animal (salamander) cells was described by Walther Flemming. in contrast to their genic contents. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. 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. concluding an XX/XO sex determination mechanism. He revised his opinion later from 46 to 48. Pre-treating cells in a hypotonic solution. von Waldeyer in 1888. in 1882. The next stage took place after the development of genetics in the early 20th century. The name was coined by another German anatomist.

It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. a find which eventually led to her Nobel Prize in 1983. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). persimilis from wild populations in California and neighboring states. Using Painter's technique they studied the polytene .6. reducing the number. 4.6 Applications in biology 4. 4. During her cytogenetic work. Human chromosome 2 was formed by a merger of ancestral chromosomes. McClintock discovered transposons. the great apes have 48 chromosomes.2 Natural populations of Drosophila In the 1930s.6. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. Rather interestingly. In 1931. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Arresting mitosis in metaphase by a solution of colchicine 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.3.

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

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

Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. 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. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations.FIG Advent of banding techniques In the late 1960s. 4.8 Beginnings of molecular cytogenetics . Deletion syndromes such as DiGeorge syndrome. Deletions within one chromosome could also now be more specifically named and understood. Caspersson developed banding techniques which differentially stain chromosomes. and elongation techniques for all culture types that allow for higher resolution banding.

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. CHAPTER 5 Techniques 5. cloned and studied in ever greater detail. movement was now made in using fluorescent labeled probes. Hybridizing them to chromosomal preparations using existing techniques came to be known as fluorescent in situ hybridization (FISH). This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely.In the 1980s. While radioisotope-labeled probes had been hybridized with DNA since 1969.1 Karyotyping .

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,

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

MATLAB eliminates the need for ‘for’ loops. such as declaring variables. You can integrate your MATLAB code with other languages and applications. 6. and allocating memory.MATLAB provides a number of features for documenting and sharing your work. It enables fast development and execution. specifying data types. one line of MATLAB code can often replace several lines of C or C++ code. The image processing step is composed of the following operations. With the MATLAB language. 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. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. In many cases.1 Image Processing The image processing step aims at image contrast enhancement and compensation of geometric distortions observed in each chromosome not related with its intrinsic shape or size. and distribute your MATLAB algorithms and applications. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. As a result. These effects must be compensated to improve the results of the pairing algorithm. .

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

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

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.'method'. . There is a large number of edge finding algorithms in existence.parameters. .2. If an image is being sent electronically from one place to another. via satellite or wireless transmission. caused by external disturbance. and we shall look at some of the more straightforward of them. Cleaning an image corrupted by noise is thus an important area of image restoration. 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.3 Two dimensional convolutions C = conv2(A. to isolate particular objects from their background. we may expect errors to occur in the image signal. to recognize or classify objects.4 Denoising We may define noise to be any degradation in the image signal.2.6. The general Matlab command for finding edges is edge(image. ) Where the parameters available depend on the method used 6. Usually we know what type of errors to expect.B) computes the two-dimensional convolution of matrices A and B. 6.5 Edge detection Edges contain some of the most useful information in an image. and hence the type of noise on the image. or through networked cable.

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

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

j).1).29.8.19.55. 36.6. end flag=0.y1)=255. y1=rc(i.22.66].31.50.9.30.10.c] = find(L==L_number((Test_number(x)))). Index=Index+1.52.40.57.7. for i=1:sx x1=rc(i. end end L_number.51. end.26. n1(x1. for x=1:46 [r.15.28.39.45.38.20.imy).21.60.32.49.59.56.24.imshow(n1. rc = [r c].35.end end if flag~=1 L_number(Index)=L(i.48.11.27. [sx sy]=size(rc).4.42.65.2). Test_number=[3.33.41.14.[]). end %h=figure. . n1=zeros(imx.43.54.62.

y)==1 Circumference_sum=Circumference_sum+1.'canny'). Arm_length_sum=0.1).end Circumference=zeros(46. for i=1:46 f=imread(strcat(num2str(i).'skel'. skel=im2double(f). end end end Circumference(i)=Circumference_sum.1).bmp')). Arm_length=zeros(46. BW1=edge(BW.Inf). BW=im2bw(f).'. Circumference_sum=0.1). s1=bwmorph(s. [m n]=size(BW1).'spur'. Area=zeros(46.5*graythresh(skel)). skel=im2bw(skel. BW=double(BW). . f=imcomplement(f). for x=1:m for y=1:n if BW1(x.1.8). s=bwmorph(skel.

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

end end end for i=1:45 if Pair(i. Pair(46.2)=i+1. Pair(i.2)==46 Pair(46. end end Pair. 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)).Area.2). 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)=i. .2)=j.1)=i. Pair(i. Pair(i. Pair=zeros(46.1)=46. Pair(i.

2).figure_flag). end end if flag~=1 if figure_flag~=47 subplot(23. end f2=imread(strcat(num2str(Pair(i. delete(figure_flag)=Pair(i. imshow(f2).bmp')).bmp')).delete=zeros(46.figure_flag). figure_flag=1. end f1=imread(strcat(num2str(Pair(i.1)==delete(j) flag=1. figure_flag=figure_flag+1. imshow(f1). if figure_flag~=47 subplot(23.2)).'.1)).'. for i=1:46 for j=1:46 if Pair(i.1). .2. figure_flag=figure_flag+1. end flag=0. flag=0.2.

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

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

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

Chapter XII: The Karyotype. 465-502. From 48 to 46: cytological technique. Res.J. 3. The material basis of heredity. 1912. ^ Stebbins G. 93. 23. ^ Concise Oxford Dictionary London.J. p. Cambridge University Press. A dictionary of genetics.L. Edinburgh. 11. Anat. Stansfield W. 1931. 6th ed. 10. 3rd ed. Arch. Animal cytology and evolution. Variation and evolution in plants. Plant Breed. 2nd ed.A. 7th ed. preconception and the counting of the human chromosomes. 9. 1939. ^ von Winiwarter H. 1922. The chromosomes. The morphology of chromosomes. State Publication Office of the Ukraine. ^ Painter T. ^ a b White M. 1973.K.. Cambridge University Press.P Oxford & NY. 4. 48. [in Russian] 6.S. and Mulligan P. Etudes sur la spermatogenese humaine. 1950. 1974.1. 1924. Oxford U. ^ Darlington C. ^ Levitsky G. 7. Bull. Evolution of genetic systems. 28 2. p242 5. Hist. Bull. revised and enlarged. 1958. 8.C.D. Columbia University Press NY. ^ King R. Med. 129. 147–49.D. ^ a b c White M. 27. 1973. 2006. Kiev.D. The spermatogenesis of man. 19-174. ^ Kottler M.A.D. . Oliver & Boyd. Chapman & Hall. Genet. ^ Levitsky G. Applied Bot. biologie 27.

p218-9 20. 1923. 13. PNAS 97. 2001. Bernard V. 1996.J. Oxford.C. & Tobler H.M. 1-6. Chromosome elimination in sciarid flies. M.R. Zoology 37. New York. 1979. p85-6 18. ^ Godfrey L. ^ Maynard Smith J.L. Arnold. 291-336. 1971.nih. The chromosome number of man. Eosin Y and Azure-A. ^ Müller F.fcgi?artid=34032 19.gov/articlerender. Chromosomal evolution in higher plants. Chromosome stains. Bioessays18: 133–138. 21. 1956.12.C. 2nd ed. NY. J. ^ Painter T. 15. Barch. The spermatogenesis of humans. and Masters J. 1998. 2000. 9821– 9823. Bioessays23: 242–250. In The ACT Cytogenetics Laboratory Manual 2nd ed.pubmedcentral.B. and Esteban M. Studies in mammalian spermatogenesis II.S.H & Levan A. ^ Goday C. The Association of Cytogenetic Technologists. Evolutionary genetics. 14. ^ A preparation which includes the dyes Methylene Blue. Chromatin diminution in nematodes. London. Human and mammalian cytogenetics: a historical perspective.R. ed. . Exp. Hereditas 42.^ a b Hsu T. ^ Tjio J.C 16. 1991.http://www. Raven Press. ^ Stebbins G. Kinetophore reproduction theory may explain rapid chromosome evolution. Springer-Verlag. 17.^ a b Gustashaw K.

Science 168. Y. Stamford CT.1007/BF02153623. 2006. Chapter 9 24.H. ^ Gilbert S. R. 8th ed. Chapman. 1990. F. Retrieved 2008-03-18. Exp. 2006.C. and Mulligan P. Experientia (Basel) 1 (2): 50– 56. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). 1970. 25. 26.F. Indian Muntjac..S. doi:10. (1945-05-15). 27.K. 2001.A. Nam. J. Stansfield W. ^ Matthey. 23. Noh.K. Sinauer Associates. Park. Chromosome evolution in the genus Ophioglossum L. Oxford U. . 1364-1366..doi:10. 291: 310–16. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods.H. ^ Khandelwal S.R..D.A. A dictionary of genetics. "L'evolution de la formule chromosomiale chez les vertébrés". Muntiacus muntjak: a deer with a low diploid number. 28. ^ King R. C. (2005).P Oxford & NY. ^ Wyngaard G.K. 7th ed. ^ Kim. ^ Wurster D.1007/s10228-004-0257-z. Developmental biology. D. Retrieved 2011-03-16. & Gregory T. and Benirschke K. J. Ichthyological Research 52 (1): 97. Botanical Journal of the Linnean Society 102: 205–217... Zool.22.

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