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

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

XY or 47. rather than 2. reflecting their bacterial origins. The individual would have Down Syndrome and his/her karyotype would be written 47.+21. If the mutation involves only one or a few chromosomes in the genome (e. in which an individual has 3.3 MUTATIONS IN CHROMOSOME NUMBER Normally.g. . An example of aneuploidy is trisomy 21.+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). 1. Euploid human karyotypes are 46.are also found in mitochondria and chloroplasts. copies of chromosome 21. a extra copy of human chromosome 21). Chromosomal Mutations are substantial changes in chromosome structure that are large enough to be visible by karyotyping (see lab manual) and thus typically affect more than one gene. Such individuals are called euploid and have the wild-type chromosome complement for the species. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins. XX (female) or 46 XY (male).XX. the individual carrying the mutation is said to be aneuploid.

chromosomes are structurally highly condensed. They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. which enables these giant DNA structures to be contained within a cell nucleus. a pair of sister chromatids attached to each other at the centromere. 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. Once the cells have divided. along with special proteins. the chromatids are uncoiled and DNA can again be transcribed. This compact form makes the individual chromosomes visible. longer-lasting attachment in this region. q-g "grande").4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division). During mitosis. The shorter arms are called p arms (from the French petit. A special DNA base sequence in the region of the kinetochores provides.Fig 1. so that each daughter cell inherits one set of chromatids. In spite of their appearance. This is the only natural context in which individual chromosomes are visible with an optical microscope. the chromatin strands become more and more condensed. and they form the classic four arm structure. The microtubules then pull the chromatids apart toward the centrosomes. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. 1.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. .

in adults weighing 65 kg (143 lbs).5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. organelles and cell membrane into two cells containing roughly equal shares of these cellular components.7 lbs). bone marrow in large bones produces new blood cells.1. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. in two separate nuclei. [1] Bone marrow is also a key component of the lymphatic system. cytoplasm. On average. It is generally followed immediately by cytokinesis. In humans. bone marrow accounts for approximately 2. bone marrow constitutes 4% of the total body mass of humans. producing the lymphocytes that support the body's immune system CHAPTER 2 2. . which use the bone marrow vasculature as a conduit to the body's systemic circulation. which divides the nuclei.6 kg (5.1 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.

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

These two cells are identical and do not differ in any way from the original parent cell. the parent cell must make a copy of each chromosome before mitosis. Each chromosome now has an identical copy of itself. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs. .Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. 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. Because each resultant daughter cell should be genetically identical to the parent cell. This occurs during the S phase of interphase.

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

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

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

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

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

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

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

forms around each set of separated sister chromosomes. however.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. unfold back into chromatin. Corresponding sister chromosomes attach at opposite ends of the cell. but cell division is not yet complete. It "cleans up" the after effects of mitosis. cytokinesis is a separate process that begins at the same time as telophase. Mitosis is complete. pinching off the separated nuclei. the nonkinetochore microtubules continue to lengthen. but rather a separate process. 2.2. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. cell . necessary for completing cell division.5 Cytokinesis Cilliate undergoing cytokinesis. In animal cells. In both animal and plant cells. A new nuclear envelope. Cytokinesis is technically not even a phase of mitosis. Both sets of chromosomes. using fragments of the parent cell's nuclear membrane. At telophase. now surrounded by new nuclei. elongating the cell even more.

2. This is the basis of the development of a multicellular body from a single cell i. Similarly.2 Development and growth The number of cells within an organism increases by mitosis. The phragmoplast is a microtubule structure typical for higher plants.5.4 Regeneration . Following are the occasions in the lives of organism where mitosis happens: 2. cells are constantly sloughed off and replaced by new ones.1Significance Mitosis is important for the maintenance of the chromosomal set. whereas some green algae use a phycoplast microtubule array during cytokinesis.. 2. New cells are formed by mitosis and so are exact copies of the cells being replaced. zygote and also the basis of the growth of a multicellular body.3 Cell replacement In some parts of body.e. Each daughter cell has a complete copy of the genome of its parent cell. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall.division is also driven by vesicles derived from the Golgi apparatus. skin and digestive tract.g. separating the two nuclei. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell.5. 2. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis. which move along microtubules to the middle of the cell. e.5.5. The end of cytokinesis marks the end of the M-phase.

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

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

carrying genetic information.7 Metaphase Metaphase. from the ancient Greek(between) and (stage). 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. 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 equally strong people. resulting in cells with many copies of the same chromosome occupying a single nucleus. an imaginary line that is equidistant from the two centrosome poles. align in the middle of the cell before being separated into each of the two daughter cells. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Preceded by events in prometaphase and followed by anaphase. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). 2.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. In certain types of cells. Early events of metaphase can . microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase. only roughly lining up along the middleline. An example of a cell that goes through endomitosis is the megakaryocyte. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes.2.

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

Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. losses of chromosomal segments or translocations. such as bcr-abl in chronic myelogenous leukemia. Malignant cells from solid tumors or leukemia samples can also be used for cytogenetic analysis to generate metaphase preparations. for example. .methods like FISH and as a hybridization matrix for comparative genomic hybridization (CGH) experiments. which may lead to chimeric oncogenes.

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

the discoverer of mitosis. which swells them and spreads the chromosomes . von Waldeyer in 1888.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. The name was coined by another German anatomist. these results were quite remarkable. The next stage took place after the development of genetics in the early 20th century. at first favoring 46. Using cells in culture 2. He revised his opinion later from 46 to 48. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. The subsequent history of the concept can be followed in the works of Darlington and White. 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.3. in contrast to their genic contents. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. in 1882. 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. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. and he correctly insisted on humans having an XX/XY system. concluding an XX/XO sex determination mechanism. Considering their techniques. Their behavior in animal (salamander) cells was described by Walther Flemming. Pretreating cells in a hypotonic solution.

Squashing the preparation on the slide forcing the chromosomes into a single plane 5.2. 3. Usually. 3. Rather interestingly.2 Observations on karyotypes 3. Human chromosome 2 was formed by a merger of ancestral chromosomes. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.2.1 Staining The study of karyotypes is made possible by staining. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture. a suitable dye. the great apes have 48 chromosomes.2 Observations Six different characteristics of karyotypes are usually observed and compared: . The sex of an unborn fetus can be determined by observation of interphase cells.3. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. [16] Sometimes observations may be made on non-dividing (interphase) cells. is applied after cells have been arrested during cell division by a solution of colchicine. Arresting mitosis in metaphase by a solution of colchicines 4. such as Giemsa. reducing the number. For humans.

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

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

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

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

The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. 14. but in grasses the average is much higher. where there are more than two sets of homologous chromosomes in the cells. about 70%. 3. 15. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. Polyploidy in animals is much less common. FN ≤ 2n. and some species of ferns have reached levels of polyploidy far in excess of the highest levels known in flowering plants. 3. Polyploidy in lower plants (ferns. and aneuploids are another example. where one sex is diploid.3 Fundamental number The fundamental number. and in some other groups. Thus. It has been of major significance in plant evolution according to Stebbins. though in this case they would not be regarded as normal members of the population. Polyploidy. 21 and 22). Haplo-diploidy. horsetails and psilotales) is also common. Humans have FN = 82.Endopolyploidy occurs when in adult differentiated . of a karyotype is the number of visible major chromosomal arms per set of chromosomes. occurs mainly in plants.means that chromosome number can vary even within one interbreeding population. It is a common arrangement in the Hymenoptera. Polyploid series in related species which consist entirely of multiples of a single basic number are known as euploid. due to the presence of five acrocentric chromosome pairs (13. FN. and the other haploid.3.4 Ploidy Ploidy is the number of complete sets of chromosomes in a cell. but it has been significant in some groups.

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

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

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

7. . human genome. late-replicating and AT rich. The pattern of bands is very similar to that seen in G-banding. • T-banding: visualize telomeres.There are other animals and plants on the Hawaiian archipelago which have undergone similar. early-replicating and GC rich. The light regions tend to be euchromatic. if less spectacular. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).7 Depiction of karyotypes 3.1 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. • Q-banding is a fluorescent pattern obtained using quinacrine for staining. This method will normally produce 300-400 bands in a normal. adaptive radiations. so it stains centromeres. R-banding is the reverse of G-banding (the R stands for "reverse"). 3. It yields a series of lightly and darkly stained bands . • • C-banding: Giemsa binds to constitutive heterochromatin.the dark regions tend to be heterochromatic.

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

Image processing software then assigns a pseudo color to each spectrally different combination. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. The critical region for this syndrome is deletion of 15. It is written as 46.XX. . Because there are a limited number of spectrally-distinct fluorophores.chromosome 5.del(5)(p15.2) 3. which is written as 46.7. allowing the visualization of the individually colored chromosomes. This technique is used to identify structural chromosome aberrations in cancer cells and other disease conditions when Giemsa banding or other techniques are not accurate enough. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.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.2. a combinatorial labeling method is used to generate many different colors.XX.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale. 3. This method is also known as virtual karyotyping.5p-. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope.

CHAPTER 4 .

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

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

segments from two different chromosomes have been exchanged. Tetrasomy. Duplications: A portion of the chromosome is duplicated. also called the terminal 11q deletion disorder. which is caused by partial deletion of the short arm of chromosome 4. In a Robertsonian translocation. • • Translocations: When a portion of one chromosome is transferred to another chromosome. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. an X. an entire chromosome has .).4. resulting in extra genetic material. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. rather than two). 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. etc. and Jacobsen syndrome. 4.3 Structural abnormalities When the chromosome's structure is altered. This can take several forms: • Deletions: A portion of the chromosome is missing or deleted. There are two main types of translocations.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). In a reciprocal translocation. Known disorders in humans include Wolf-Hirschhorn syndrome.

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

at first favoring 46. The next stage took place after the development of genetics in the early 20th century. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. in 1882. Their behavior in animal (salamander) cells was described by Walther Flemming. Painter in 1922 was not certain whether the diploid number of man was 46 or 48.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. He revised his opinion later from 46 to 48. Pre-treating cells in a hypotonic solution. New techniques were needed to definitively solve the problem: 1. these results were quite remarkable. 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.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). the discoverer of mitosis. which swells them and spreads the chromosomes . and he correctly insisted on man having an XX/XY system. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Using cells in culture 2. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. The name was coined by another German anatomist. in contrast to their genic contents. Considering their techniques. concluding an XX/XO sex determination mechanism. 4. von Waldeyer in 1888.

4. McClintock discovered transposons. reducing the number. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. the great apes have 48 chromosomes. Cutting up a photomicrograph and arranging the result into an indisputable karyogram. Using Painter's technique they studied the polytene . a find which eventually led to her Nobel Prize in 1983. During her cytogenetic work. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize.2 Natural populations of Drosophila In the 1930s. Rather interestingly. 4. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). Human chromosome 2 was formed by a merger of ancestral chromosomes.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.6. persimilis from wild populations in California and neighboring states.6. Squashing the preparation on the slide forcing the chromosomes into a single plane 5.3. Arresting mitosis in metaphase by a solution of colchicine 4.6 Applications in biology 4. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. In 1931.

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

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

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

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

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,

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

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

2) Geometrical compensation—The geometric compensation. a dimensional scaling is performed before the pattern features is extracted to make all the chromosome with the same size and aspect ratio by interpolating the original images. or at least attenuated.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast.2 Concepts used in this phase 1) Image conversion 2) Denoising . To compensate for this inhomogeneity. This compensation algorithm is composed of the following main steps: a) chromosome and medial axis segmentation b) axis smoothing c) interpolationalong orthogonal lines to the smoothed medial axis d) border regularization 3) Shape normalization—The features used in the comparison of chromosomes are grouped into two classes: 1) geometric based 2) pattern based (G-banding). the spatially scaled images are histogram equalized. geometrical and dimensional differences must be removed. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. Therefore. 6. To compare chromosomes from a band pattern point of view.

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

.'method'. to isolate particular objects from their background.6. The general Matlab command for finding edges is edge(image.parameters. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. ) Where the parameters available depend on the method used 6. and hence the type of noise on the image. If an image is being sent electronically from one place to another.3 Two dimensional convolutions C = conv2(A. to recognize or classify objects. There is a large number of edge finding algorithms in existence.2. . via satellite or wireless transmission. If one of these matrices describes a two-dimensional finite impulse response . hence we can choose the most appropriate method for reducing the effects. we may expect errors to occur in the image signal. and we shall look at some of the more straightforward of them. 6. caused by external disturbance.B) computes the two-dimensional convolution of matrices A and B. Usually we know what type of errors to expect.4 Denoising We may define noise to be any degradation in the image signal. We may use edges to measure the size of objects in an image.2. or through networked cable.5 Edge detection Edges contain some of the most useful information in an image. Cleaning an image corrupted by noise is thus an important area of image restoration.

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

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

31.60.30.11.26.c] = find(L==L_number((Test_number(x)))).end end if flag~=1 L_number(Index)=L(i.38.27.39.59.[]).y1)=255. [sx sy]=size(rc).66].55. end end L_number. for i=1:sx x1=rc(i.51.32.7.52. y1=rc(i.10.8.9. rc = [r c]. Test_number=[3.imy). end %h=figure.28.14.43.19.42.4. n1(x1.54.29.50.41.21.35.1). . for x=1:46 [r.48.40.65.6.imshow(n1.j).45.22. Index=Index+1.15. n1=zeros(imx.49. 36.57.62.33.24.56.2).20. end flag=0. end.

skel=im2double(f). BW=im2bw(f).'.bmp')).1). .'canny').y)==1 Circumference_sum=Circumference_sum+1.'spur'.8).'skel'.end Circumference=zeros(46. Arm_length_sum=0. BW=double(BW). Circumference_sum=0.1). Arm_length=zeros(46.1).5*graythresh(skel)). Area=zeros(46. [m n]=size(BW1).1. skel=im2bw(skel. for x=1:m for y=1:n if BW1(x. for i=1:46 f=imread(strcat(num2str(i). end end end Circumference(i)=Circumference_sum.Inf). BW1=edge(BW. f=imcomplement(f). s=bwmorph(skel. s1=bwmorph(s.

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

2)=i+1. Pair(i.2)=i.2)==46 Pair(46.1)=46. Pair(46.2). Pair(i.1)=i. Pair(i. 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)).Area.1)=i. . Pair=zeros(46. for j=1:46 if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j))<min min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)Arm_length(j))+abs(Area(i)-Area(j)).2)=j. Pair(i. end end Pair. end end end for i=1:45 if Pair(i.

1).2)).bmp')).bmp')). figure_flag=1.'. figure_flag=figure_flag+1.figure_flag). imshow(f2). delete(figure_flag)=Pair(i.2. imshow(f1). if figure_flag~=47 subplot(23. end flag=0.1)==delete(j) flag=1.2. end f1=imread(strcat(num2str(Pair(i.delete=zeros(46. flag=0.2).1)). end f2=imread(strcat(num2str(Pair(i. figure_flag=figure_flag+1.'.figure_flag). end end if flag~=1 if figure_flag~=47 subplot(23. for i=1:46 for j=1:46 if Pair(i. .

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

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

The results presented in this paper are promising. a new chromosome dataset with 9200 chromosomes from bone marrow cells. Using 27 karyograms andworking with a limited number of classes (≤ 8). REFERENCES .performance of the classifier. presenting a uniform level of condensation. called LK1 . This dataset was made publicly available [29]. Executing the algorithm on a higher quality dataset. and from which it is possible to extract additional features. a 76.10% classification ratewas obtained. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. or Philadelphia. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes.. amean classification rate larger than 93% was obtained in all experiments. centromere position. Copenhagen. whose images are of significantly higher quality. despite the low quality of this type of chromosomes. e. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements.g. In addition. In fact. such as Edinburgh.

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A. 2001. Nam. ^ Wyngaard G. Chapter 9 24.P Oxford & NY. ^ Matthey. Chromosome evolution in the genus Ophioglossum L. Indian Muntjac. & Gregory T.doi:10. Exp. Oxford U. 1990. Botanical Journal of the Linnean Society 102: 205–217.K.K.R. ^ Gilbert S.F. Stamford CT. 1364-1366.A. Retrieved 2008-03-18. 2006. C. D.S. Temporal control of DNA replication and the adaptive value of chromatin diminution in copepods. 8th ed. Zool. (1945-05-15). Stansfield W..H. 23. Ichthyological Research 52 (1): 97. ^ Kim. ^ Wurster D. ^ King R..C. R. Noh.K. "Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the 94– Acipenseriformes" (PDF). Retrieved 2011-03-16. 27. Experientia (Basel) 1 (2): 50– 56. 291: 310–16. 28. Y. and Benirschke K. Developmental biology. . and Mulligan P.H. (2005). Sinauer Associates. Muntiacus muntjak: a deer with a low diploid number. 26. doi:10. J. Park... Chapman. Science 168. 25. F.D. A dictionary of genetics. ^ Khandelwal S. 2006. "L'evolution de la formule chromosomiale chez les vertébrés". J.22.1007/s10228-004-0257-z.. 7th ed. 1970.1007/BF02153623.

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